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Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent... Purinergic Signalling (2006) 2:451–469 DOI 10.1007/s11302-006-9008-0 Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks Kristof Van Kolen & Herman Slegers Received: 19 July 2005 / Accepted: 17 March 2006 / Published online: 7 June 2006 # Springer Science + Business Media B.V. 2006 Abstract The role of nucleotides in intracellular energy Abbreviations provision and nucleic acid synthesis has been known for AC adenylate cyclase 1 3 a long time. In the past decade, evidence has been ApAP ,P -di(adenosine-5 )triphosphate 1 4 presented that, in addition to these functions, nucleo- ApAP ,P -di(adenosine-5 )tetraphosphate tides are also autocrine and paracrine messenger AR adrenergic receptor molecules that initiate and regulate a large number of COX cyclooxygenase biological processes. The actions of extracellular nucle- DAG diacylglycerol otides are mediated by ionotropic P2X and metabo- ERK extracellular signal-regulated kinase tropic P2Y receptors, while hydrolysis by ecto-enzymes GFAP glial fibrillary acidic protein modulates the initial signal. An increasing number of GPCR G protein-coupled receptor studies have been performed to obtain information on HT hydroxytryptamine the signal transduction pathways activated by nucleo- IP inositol (1,4,5)-triphosphate 0 0 tide receptors. The development of specific and stable PAP adenosine-3 ,5 -biphosphate purinergic receptor agonists and antagonists with ther- PI phosphatidylinositol apeutical potential largely contributed to the identifi- PL phospholipase cation of receptors responsible for nucleotide-activated PI 3-K phosphatidylinositol 3-kinase pathways. This article reviews the signal transduction PPADS pyridoxalphosphate-6-azophenyl-2 , pathways activated by P2Y receptors, the involved 4 -disulphonate second messenger systems, GTPases and protein Pyk2 proline-rich tyrosine kinase 2 kinases, as well as recent findings concerning P2Y RKIP Raf kinase inhibitory protein receptor signalling in C6 glioma cells. Besides vertical RTK receptor tyrosine kinase signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed. Introduction . . . . Key words C6 glioma ERK P2Y receptors PKB Pharmacological properties of P2Y receptors transactivation tyrosine kinases Extracellular actions of adenine nucleotides were ini- tially characterised in the cardiovascular system by Drury and Szent-Gyorgyi [1]. It took more than four K. Van Kolen H. Slegers (*) Department of Biomedical Sciences, Cellular Biochemistry, decades before the concept of purinergic signalling was University of Antwerp, accepted, but now it is well established that nucleo- Universiteitsplein 1, tides initiate and regulate a variety of biological 2610 Wilrijk-Antwerpen, Belgium processes, including neurotransmission, inflammation, e-mail: herman.slegers@ua.ac.be regulation of blood pressure, platelet aggregation, cell K. Van Kolen growth and differentiation (Abbracchio et al. [2]; CNS research, Johnson & Johnson, PRD, Burnstock and Williams [3]; Burnstock [4]; Ralevic Janssen Pharmaceutica, and Burnstock [5]). Beerse, Belgium 452 Purinergic Signalling (2006) 2:451–469 Nucleotides are released in the extracellular fluid by tides (P2Y ). The pharmacological profile of the cell lysis, exocytosis, secretion of granules, efflux and recently cloned P2Y receptor is distinct from the upon cellular stress such as changes in osmolarity and other P2Y receptors since UDP-glucose, UDP-galac- mechanical perturbations. Once released, they mediate tose, UDP-glucuronic acid and UDP-N-acetylglucos- their effect by stimulation of nucleotide receptors. amine are specific ligands of this receptor (Chambers Based on pharmacological properties, the first sug- et al. [13]). Natural P2Y receptor ligands do not gestion for the existence of ionotropic P2X receptors exclusively bind to one receptor subtype. ADP is an and metabotropic P2Y receptors was made by agonist of P2Y ,P2Y and P2Y receptors, whereas 1 12 13 Burnstock and Kennedy [6]. After cloning, multiple ATP is a full agonist of P2Y and P2Y ,but a partial 2 11 subtypes of P2X and P2Y receptors were characterised agonist or antagonist of P2Y ,P2Y and P2Y 1 12 13 unambiguously (Abbracchio and Burnstock [7] receptors. Although the pharmacological properties of Burnstock and Williams [3]; Fredholm et al. [8]). P2Y receptors (Table 1) are well conserved between Up to now, the P2Y receptor family comprises at least species, some remarkable differences have been ob- eight subtypes, P2Y ,P2Y ,P2Y ,P2Y ,P2Y ,P2Y , served. While UTP acts as an agonist of both human and 1 2 4 6 11 12 P2Y and the recently identified P2Y receptor rat P2Y receptors, ATP is a potent agonist of the rat 13 14 4 (Ralevic and Burnstock, [5] Abbracchio et al. [9]; P2Y , but an antagonist of the human orthologue. Communi et al. [10]; Hollopeter et al. [11]; Zhang Mutational analysis revealed that the second extracel- et al. [12]). According to the agonist profile, P2Y lular loop of the P2Y receptor is responsible for the receptors can be subdivided into receptors responding opposing effect of ATP in both species (Herold et al. to adenine mono- and dinucleotides (P2Y , P2Y , [14]). A similar phenomenon is observed when human 1 11 P2Y ,P2Y ), and to uridine nucleotides (P2Y , and canine P2Y receptors were stably expressed in 12 13 4 11 P2Y ), and receptors for adenine and uridine nucleo- CHO-K1 and 1321N1 astrocytoma cells. Whereas the Table 1 Pharmacological profile of P2Y receptors and second messenger systems. Agonists Antagonists Effector G protein P2Y 2MeSADP, ADP, ADP"S, Suramin, PPADS, PAP, MRS2179, MRS2216, PLC, I G /G 1 K, Ca q 11/12 Ap A, MRS2365 MRS2279, MRS2500, MRS 2603 P2Y UTP+S, ATP+S, UTP, ATP, Suramin PLC, I G /G 2 K, Ca i q INS37217, Ap A a a P2Y UTP+S, UTP, ATP ATP , PPADS PLC, I G /G 4 K q 11/12 P2Y UDP"S, UDP, UTP, INS48823 PPADS, suramin, MRS2567 PLC, I G /G 6 K q 11/12 P2Y ATP!S, ATP+S, ATP Suramin AC, PLC G /G 11 q s P2Y 2MeSADP, ADP, Ap A, ATP, AR-C69931MX, AR-C67085, AR-C78511KF, AC, I G 12 3 K, Ca i/o Ap A clopidogrel, 2MeSAMP, DIDS, suramin, MRS2395 P2Y 2MeSADP, ADP, Ap A, ATP AR-C69931MX, Ap A, PPADS, suramin, MRS2211, AC, PLC, I G /G 13 3 4 Ca i q MRS2603 P2Y UDP-glucose, UDP-galactose, AC, I G 14 Ca i UDP-glucuronic acid, UDP-N-acetylglucosamine ATP acts as an agonist of the rat P2Y but as an antagonist of acid monoammonium salt; DIDS, 4,4 -diisothiocyanatostilbene-2, 0 0 0 the human P2Y receptor (Herold et al [14]). Reactive blue 2 is not 2 -disulphonic acid; INS37217 [P(1)-(uridine 5 )-P (4)-(2 -deoxy- included in the list since it displays lack of specificity towards the cytidine 5 )tetraphosphate tetrasodium salt; INS48823 P -((2-benzyl-1, 3 6 0 0 different P2Y subtypes. References: Abbracchio et al. [9]; Com- 3-dioxolo-4-yl)uridine 5 )P -(uridine 5 ) triphosphate; MRS2179, N - 0 0 0 0 muni et al. [10, 191]; Chambers et al. [13]; Claes and Slegers [17]; methyl-2 -deoxyadenosine-3 ,5 -bisphosphate; MRS2211, pyridoxal- 5 - Kim et al. [26]; Xu et al. [27]; Boyer et al. [38, 189, 190]; Grobben phosphate-6-azo-(2-chloro-5-nitrophenyl)-2,4-disulphonate; MRS2216, 0 0 0 et al. [40]; Marteau et al. [47]; Filippov et al. [57– 60, 63]; Simon 2 -deoxy-2-chloro-N -methyladenosine-3 ,5 -bisphosphate; MRS2279, 0 0 0 et al. [61]; Wirkner et al. [62]; Korcok et al. [192]; Muller [193]; 2-chloro-N -methyl-(N)-methanocarba-2 -deoxyadenosine 3 ,5 -bis- 0 0 0 0 0 Skelton et al. [194]; Yerxa et al. [195]; Jacobson et al. [196]; von phosphate; MRS2365, [(1 S,2 R,3 S,4 R,5 S)-4-[(6-amino-2-methylthio- 1 3 Ku¨ gelgen [197]. Abbreviations: Ap A, P ,P -di(adenosine-5 ) 9H-purin-9-yl)-1-diphosphoryloxymethyl]bicyclo[3.1.0]hexane-2, 3-diol]; 1 4 triphosphate; Ap A, P ,P -di(adenosine-5 )tetraphosphate; AR- MRS2395, 2-dimethyl-propionic acid-3-(2-chloro-6-methylaminopurin- C69931MX, N -(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio) 9-yl)-2-(2,2-dimethylpropionyloxy-methyl)-propylester; MRS2500, 2-io- 0 0 0 -",+-dichloromethylene ATP; AR-C67085, 2-propylthio-D-"+- do-N -methyl-(N)-methanocarba-2 -deoxyadenosine 3 ,5 -bisphosphate; dichloromethylene adenosine 5 -triphosphate; AR-C78511KF, MRS2567, 1,2-di-(4-isothiocyanatophenyl)ethane; MRS2603, pyridox- (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyri- al-5 -phosphate-6-azo-(4-chloro-3-nitrophenyl)-2,4-disulphonate; 0 0 midin-3-yl]-1,5,6-trideoxy-"-D-ribo-hept-5-enofuranuronoyl]-L-aspartic PPADS, pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid. Purinergic Signalling (2006) 2:451–469 453 human P2Y is potently activated by adenosine tri- cAMP (dBcAMP) or 8-chloro-cAMP, induces differ- phosphate nucleotides, the canine orthologue displayed entiation into an astrocyte type II (Roymans et al. more selectivity towards the corresponding diphos- [34]). During this process, cessation of cell growth is phates. In this case, the nucleotide selectivity is due to accompanied by a shift in intermediate filament differences in the amino acid sequence at the juxtapo- synthesis from vimentin to glial fibrillary acidic protein sition of TM6 and the third extracellular loop also (GFAP) (Backhovens et al. [35]). The latter protein is reported to play an important role in agonist selectivity an astrocytic differentiation marker whose expression and signalling of other G protein-coupled receptors is regulated by cAMP at the transcriptional and (GPCR) (Qi et al. [15]; Lawson and Wheatley [16]). translational level (Messens and Slegers [36]). Despite their chemical stability, extracellular nu- In our laboratory, the signalling pathways activated by cleotides are metabolised by several ecto-enzymes extracellular nucleotides, and in particular those affecting (Claes and Slegers [17]; Czajkowski and Baranska cell proliferation and differentiation of C6 cells, were [18]; Goding et al. [19]; Zimmerman [20]). Extracellu- studied in detail. The presence of a P2Y receptor on these lar hydrolysis complicates the evaluation of nucleo- cells that negatively affects adenylate cyclase (AC) was tide-mediated effects on different cell types and can be postulated for more than a decade (Pianet et al. [37]; Boyer overcome by the use of specific non-hydrolysable et al. [38]). This receptor is coupled to a G protein and receptor agonists or ecto-enzyme inhibitors. Some has been denominated P2Y - (Claes et al. [39]; Grobben AC P2Y receptor antagonists, such as pyridoxalphosphate- et al. [40]) before its identification as the P2Y receptor 0 0 6-azophenyl-2 ,4 -disulfonic acid (PPADS), suramin, initially cloned from blood platelets (Czajkowski et al. 0 0 reactive blue 2 and 4,4 -diisothiocyanatostilbene-2,2 - [41]; Hollopeter et al. [11]; Jin et al. [42]). C6 cells also disulphonic acid (DIDS), are inhibitors of nucleotide express the phospholipase (PL)C"-coupled P2Y ,P2Y , 1 2 hydrolyzing enzymes and are often used in studies of P2Y and P2Y receptors (Czajkowski et al. [41]; Nicholas 4 6 nucleotide-mediated signalling (Grobben et al. [21]). et al. [43]; Tu et al. [44]; Claes and Slegers [17]). Recently, Nevertheless, care must be taken for the interpretation we also demonstrated the presence of P2Y mRNA (Van of experimental data since it is also shown that cells can Kolen and Slegers [45]) implicating the expression of internalise some of these molecules (Claes et al., [22]). three ADP-activated receptors in these cells, i.e., P2Y 1, Therefore specific P2Y receptor antagonists, devel- P2Y and P2Y . Although 2MeSADP is reported as a 12 13 oped for therapeutical purposes, have to be used to potent P2Y ,P2Y and P2Y agonist, stimulation with 1 12 13 overcome the lack of specificity (Boeynaems et al. this compound inhibits AC, but induces no significant [23]; Lambrecht et al. [24]; Kam and Nethery [25]; Kim activation of PLC, indicating that the P2Y receptor is not et al. [26]; Xu et al. [27]). activated by ADP in cells grown in chemically defined medium (Grobben et al. [40]). This is confirmed by Czajkowski et al. [46], who showed that, in cells cultivated in the presence of fetal calf serum, ADP signalling is P2Y receptor expression on rat C6 glioma cells predominantly determined by the P2Y receptor. Howev- er, upon serum deprivation, expression of the P2Y Rat C6 glioma is a tumoral cell line of glial origin with receptor is decreased and the P2Y receptor becomes oligodendrocytic, astrocytic and neuronal progenitor the main activated receptor. Characterization of P2Y properties. Due to a point mutation in Fphosphatase receptor function is complicated by the fact that P2Y and tensin homologue deleted on chromosome ten_ and P2Y receptors have almost the same agonist profile (PTEN), the phosphatidylinositol 3-kinase (PI 3-K)/ (Table 1). The receptor antagonist N -(2-methyl- PKB signalling pathway is constitutively active and thioethyl)-2-(3,3,3-trifluoropropylthio)-",+-dichloromethy- contributes to the proliferative and invasive properties lene ATP (AR-C69931MX), often used as a specific of these cells (Kubiatowski et al. [28]; Roymans and P2Y antagonist, also blocks the P2Y receptor (Marteau 12 13 Slegers [29]; Grobben et al. [30]). In addition, cell et al. [47]). While the human and mouse P2Y receptor, proliferation is sustained by secreted growth factors like the P2Y , is more potently activated by 2MeSADP that stimulate growth factor receptors present on these than ADP, the rat P2Y receptor shows a higher cells. Such autocrine mechanisms are reported for selectivity for ADP (Fumagalli et al. [48]). In C6 cells, IGFR, bFGFR and PDGFR (Okumura et al. [31]; further distinction between the signalling of P2Y and Resnicoff et al. [32]; Strawn et al. [33]). P2Y receptors can be made by the use of PPADS, a 1 4 In C6 cells, an increase in cAMP by stimulation of P2Y antagonist without effect on P2Y ,and P ,P - 13 12 the "-adrenergic receptor ("-AR) or by addition of di(adenosine-5 ) tetraphosphate (Ap A), a P2Y antago- 4 13 membrane permeable cAMP analogues, e.g., dibutyryl nist that stimulates the P2Y receptor (Claes et al. [39]; 12 454 Purinergic Signalling (2006) 2:451–469 Grobben et al. [40]; Marteau et al. [47]). In addition, sitol 4,5-bisphosphate into the intracellular messenger recently synthesised PPADS derivatives pyridoxal-5 - inositol 1,4,5-triphosphate (IP ) and diacylglycerol phosphate-6-azo-(2-chloro-5-nitrophenyl)-2,4-disulphonate (DAG). Activation of PLC occurs by G !- and/or (MRS2211) and pyridoxal-5 -phosphate-6-azo-(4-chloro-3- G !-dependent mechanisms (Communi et al. [55]). nitrophenyl)-2,4-disulphonate (MRS2603) have no effect Besides signalling through G! subunits, intracellular 2+ on the P2Y receptor but antagonise the P2Y receptor Ca concentration is also affected by G"+ subunit- 12 13 2+ (Kim et al. [26]). dependent interaction with voltage-gated Ca chan- Although P2Y receptor expression in C6 cells de- nels. Several reports indicated modulation of K pends on the cultivation conditions (Czajkowski et al. currents and PLC" activation induced by distinct [46]), unpublished data of our laboratory revealed that domains of G"+ (Mirshahi et al. [56]). Co-expression induction of differentiation into astrocytes type II by studies performed in rat sympathetic neurons demon- dbcAMP (1 mM)- or (-)-isoproterenol (5 2M) does not strated that P2Y , P2Y and P2Y receptors trigger the 1 2 6 2+ + significantly alter the expression of P2Y receptors. closing of N-type Ca and M-type K channels, These observations are in accordance with previous whereas P2Y receptor stimulation also displayed studies on the expression of P2Y , P2Y , P2Y , P2Y , coupling to M-type K channels producing a less 1 2 4 6 2+ P2Y ,P2Y and P2Y receptors in glial cells and efficient inhibition of Ca currents. In rat brain cap- 12 13 14 primary astrocytes (Bianco et al. [49]; Fumagalli et al. illary endothelial cells, it was shown that the P2Y [48, 50]; Sasaki et al. [51]). The function of P2Y receptor receptor inhibits I and activates a G protein- Ca(N) expression in glial cells is still under investigation, but a coupled inward rectifier K (GIRK) channel. Interest- number of studies point to an important role in the ingly, stimulation of the P2Y receptor also induces a intercellular communication between astrocytes and K current that is rapidly followed by inactivation. neurons (Bezzi and Volterra, [52]). Another well Inhibition of I by P2Y receptor stimulation is Ca(N) 12 documented effect of extracellular ATP is induction also reported in PC12 cells while in HEK 293 this of reactive astrogliosis upon activation of ERK and inhibition is mediated by the P2Y receptor (Filippov cyclooxygenase (COX)-2 (Brambilla et al. [53]). et al. [57–60]; Simon et al. [61]; Wirkner et al. [62]). Similar to the observations made in C6 cells, From co-expression studies of P2Y receptors with functional responses of P2Y receptor subtypes in GIRK1 or GIRK2 in rat sympathetic neurons, it was microglial cells depend on cultivation conditions. In concluded that P2Y receptors activate GIRK channels N9 mouse brain microglia stimulation of expressed P2Y by the "+ subunits of G and inhibit these channels by i/o 2+ receptors induces Ca mobilization but only P2Y and the ! subunits of G (Filippov et al. [63]). 6 q P2Y receptor-mediated responses are increased upon P2Y-induced calcium release is followed by opening 2+ activation of microglia with lipopolysaccharide. The of voltage-independent Ca channels. Although this enhanced P2Y response is correlated with mRNA response is observed in a variety of cell types, the increase which was not the case for the P2Y physiological implications are miscellaneous. In this 2+ receptor-mediated Ca mobilization (Bianco et al. context, it has been reported that extracellular ATP 2+ [49]). Furthermore, stimulation of microglial P2Y induces a Ca wave that propagates through neigh- 12/13 receptors induces membrane ruffling and chemotaxis bouring astrocytes by GAP junctions (Suadicani et al. towards injured neurons through G protein-medi- [64]). In situations of increased neuronal activity or cell i/o 2+ ated activation of Rac (Honda et al. [54]). The damage, ATP stimulates a Ca -dependent proton- observations made in astrocytes and microglial cells efflux from astrocytes. Acidification of the extracellular emphasise the importance of P2Y receptors in brain environment serves as a negative feedback mechanism signalling and identify these receptors as putative for neurotransmitter release, but also increases blood targets in defective neurotransmission, neuroimmune flow by vasodilatation in cerebral arterioles (Dienel and 2+ functioning, cell survival and cell proliferation in Hertz [65]; Dixon et al. [66]). Although Ca signalling response to oxidative stress and brain injury. is observed in a variety of cell types, the time dependence of the response is cell type specific. This is especially the case for the P2Y receptor which 2+ P2Y receptor-activated signalling cascades triggers persistent or transient Ca responses when it is expressed in human 1321N1 astrocytoma or C6 glioma Second messengers cells, respectively (Czajkowski et al. [41]; Sellers et al. [67]). A recent study also revealed that, in glial cells, 2+ P2Y receptors are generally linked to PLC activation prolongation of the P2Y receptor-induced Ca re- + + that catalyses the rapid hydrolysis of phosphatidylino- sponse is regulated by interaction with the Na /H Purinergic Signalling (2006) 2:451–469 455 exchanger regulatory factor type-2 which determines ceptors is sometimes sufficient to exert a significant the signalling pathways that are ultimately activated in inhibitory action towards PKA. Such a response is different cell types (Fam et al. [68]). Indeed, while reported in microglial cells where ATP and ADP transient P2Y receptor signalling increases prolifera- binding to P2Y receptors mediate chemotaxis by 1 12/13 tion in C6 cells, sustained signalling triggers apoptotic PKA-dependent translocation of "1 integrins to ruf- cascades in 1321N1 astrocytoma cells (Czajkowski et al. fling regions of the cell (Nasu-Tada et al. [76]). [46]; Sellers et al. [67]). Despite the fact that cells express a myriad of A well-known response to PLC-generated DAG and different GPCRs and downstream acting regulators, 2+ IP /Ca is the activation of classical PKCs that are receptor stimulation promotes rapid and specific involved in rapid internalisation and desensitisation of responses. In addition, multiple GPCRs, sharing the GPCRs through phosphorylation of residues localised same second messenger cascade, can induce different in their cytoplasmic tail. In this regard, PKC"Iis cellular events in one cell type indicating that GPCR reported to attenuate phosphatidylinositol (PI)-hydro- signal propagation requires physical interactions in a lysis induced by P2Y and P2Y receptors in endothelial defined cellular compartment. An example of spatial 1 2 cells (Chen and Lin [69]). In astrocytes, high frequency organised signalling is the "-arrestin-dependent target- stimulation of the P2Y receptor by repeated addition ing of an activated receptor into clathrin-coated vesicles of ATP causes rapid suppression of the P2Y receptor- or enrichment in membrane microdomains (lipid rafts) 2+ induced Ca response. This phenomenon, observed as formed by cholesterol and sphingolipids (Anderson 2+ Ca oscillations, is mediated by protein kinase C- [77]; DeFea et al. [78]). Modulation of receptor dependent phosphorylation of Thr339 in the carboxy- function by rafts is confirmed for an increasing number terminus of the P2Y receptor (Fam et al. [70]). Besides of GPCRs including P2Y receptors (Anderson [77]; modulation of receptor responsiveness, PKC signalling Ostrom and Insel [79]). In endothelial cells, it is also affects long term effects. In the human osteoblastic reported that P2Y receptor-induced vasodilatation is HOBIT cell line, ATP increases expression of the early abolished by disruption of caveolae with methyl-"- growth response protein-1 by a mechanism that requires cyclodextrin (Kaiser et al. [80]). In C6 glioma cells, 2+ aCa -independent PKC isoform (Pines et al. [71]). In signalling by P2Y and 5-hydroxytryptamine (HT) 2 2A vascular smooth muscle cells, UDP stimulates cell cycle receptors is attenuated after knock-down of caveolin-1 progression by a PLC- and PKC%-dependent cascade by si-RNA. Moreover, interaction between the 5-HT 2A (Hou et al. [72]). The same isoform is involved in ATP- receptor and caveolin-1 facilitates its interaction with mediated mitogenic signalling in astrocytes, but in these G! . Since P2Y receptor mRNA is downregulated by q 2 cells PKC% activation does not involve PLC but caveolin-1 knock-down, further studies are required to requires PLD-dependent choline formation (Neary demonstrate localisation of P2Y receptors in caveolae et al. [73]). of C6 cells (Bhatnagar et al. [81]). In addition to PLC-coupled receptors, a growing number of P2Y receptors have been shown to affect the activity of AC. Besides the existence of indirect Small GTPases as molecular switches mechanisms linked to an increase in cAMP (discussed in Communi et al. [55]), only the P2Y receptor is The processing of extracellular stimuli by GPCRs often involves signalling by second messengers directly coupled to activation of AC and PLC while 2+ P2Y , P2Y and P2Y receptors negatively affect (cAMP, DAG, Ca ) towards small GTPases and/or 12 13 14 cross-talk with tyrosine kinases. G protein-coupled cAMP synthesis (Chambers et al. [13]; Communi et al. [10]; Hollopeter et al. [11]; Zhang et al. [12]). receptor signalling via PLC" induces formation of 2+ DAG and IP ,Ca mobilisation and activation of Adenylate cyclase-dependent signalling is often me- diated by the cAMP-regulated kinase PKA. Stimula- PKC ultimately leading to activation of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 cooperates with Src to tion of the P2Y receptor with ATP is shown to activate human monocyte-derived dendritic cells by recruite Grb2 and SOS, a guanine nucleotide exchange increased cAMP/PKA signalling (Wilkin et al. [74]). In factor (GEF) that activates Ras (Lev et al. [82]). Such a mechanism is reported for Ras-dependent ERK acti- bovine adrenocortical fasciculate cells, ADP and ATP increase cortisol production through PKA activation vation induced by the protease-activated receptor-1 in astrocytes (Wang and Reiser [83]). In PC12 cells, by an as yet unidentified G protein-coupled P2Y receptor (Nishi et al. [75]). Although in unstimulated stimulation of the P2Y receptor also triggers tyrosine phosphorylation of Pyk2, but further signalling to Ras cells the cytosolic cAMP concentration is already low, its further decrease by G protein-coupled re- involves EGFR transactivation by Src (Soltoff et al. i 456 Purinergic Signalling (2006) 2:451–469 [84]). Tyrosine kinase-dependent Ras signalling is also Honda et al. [54]). Stress fibre formation in vascular reported for G protein-coupled receptors, but this smooth muscle cells is reported to be mediated by proceeds through G"+ subunit-mediated activation of RhoA/ROCK signalling that becomes activated upon PI 3-K+ and Shc (Ellis et al. [85]; Lopez-Ilasaca et al. stimulation of P2Y ,P2Y , P2Y and P2Y receptors 1 2 4 6 [86]). (Sauzeau et al. [99]). In the latter study, information GEFs can also be regulated in a tyrosine kinase- concerning the signalling towards RhoA is lacking. In independent manner that proceeds through direct a more recent study on endothelial cells, transactiva- 2+ activation by cAMP, DAG and Ca or by interaction tion of VEGFR upon P2Y receptor stimulation and with G! subunits as observed for the G protein- recruitment of the RhoGEF Vav is shown to be a q/11 mediated activation of RhoA (Bhattacharya et al. [87]; possible mechanism to initiate RhoA-mediated cell Bos [88]; Lutz et al. [89]; Walker et al. [90]). adhesion (Seye et al. [100]). P2Y receptor signalling towards GTPases is involved Although several P2Y receptors activate RhoA, in short term responses, such as stress fibre formation or downstream signalling and physiological consequences modulation of cell adhesion, but also in long term are determined by celltype specific mechanisms lead- responses like cell proliferation. Mitogenic Ras-depen- ing to diverse responses. dent P2Y responses are reported for C6 and HEK293 cells where Ras is implicated in P2Y receptor-depen- ERK signalling dent signalling to the ERK pathway (Gao et al. [91]; Tu et al. [44]). On the other hand, increased prolifer- Several GPCRs are coupled to enhanced proliferation ation of C6 cells by the P2Y receptor proceeds by multiple signal transduction pathways that phos- independently of Ras, but requires RhoA-dependent phorylate ERK. Activation of this kinase requires Ras activation of ERK and Rho-associated coiled-coil- or GTPases of the RhoA family and is often modulated containing protein kinase (ROCK) (Grobben et al. by second messenger-activated pathways, although [40]; Van Kolen and Slegers, unpublished data). cross-talk with growth factor receptors also triggers Interestingly, when the P2Y receptor is expressed ERK signalling. in CHO cells it activates ERK and RhoA/ROCK In neurons G protein-mediated activation of AC by independent mechanisms (Soulet et al. [92]). increases ERK phosphorylation by a PKA/Rap1/B- Another example of cross-talk between P2Y recep- Raf cascade. In contrast, induction of cAMP synthesis tors and GTPases is observed in blood platelets. As decreases ERK phosphorylation in C6 cells and mentioned above, release of ADP and subsequent P2Y astrocytes by a negative action of PKA on the Ras/c- and P2Y receptor binding is essential for collagen- Raf1 interaction, or by Rap1-mediated inhibition of c- induced platelet aggregation. A crucial step for imme- Raf1. These observations led to the hypothesis that an diate and sustained aggregation of platelets is the increase in cAMP stimulates MEK/ERK signalling in activation of Rap1 that increases the affinity between B-Raf expressing cells but inhibits this cascade in B- integrin ! " and fibrinogen. Knock-out studies Raf negative cells (Dugan et al. [101]). In both cases, IIb 3 revealed that ADP-induced GTP loading of Rap1 PKA activation has a central role and mediates its proceeds through both G and G signalling by P2Y effects through Src and Rap1 activation (Stork and i q 12 and P2Y receptors, respectively. The mechanism Schmitt [102]). Although the majority of cAMP- initiated by the P2Y receptor is shown to be PI 3- dependent effects can be explained by this hypothesis, K-dependent while P2Y -mediated activation of Rap1 a few exceptions are reported. In some B-Raf positive 2+ requires Ca mobilisation (Woulfe et al. [93]; Greco cells, an increase in cAMP is shown to inhibit B-Raf, et al. [94]; Larson et al. [95]; Lova et al. [96, 97]). suggesting that regulation of this kinase by cAMP also Stimulation of the P2Y receptor also contributes to depends on other cell type specific factors. One model 2+ platelet shape changes by a Ca -independent path- suggests the involvement of 14-3-3 proteins acting as way. RhoA and its effector ROCK are activated by scaffolding proteins to shield B-Raf and Raf1 from ADP through G protein-dependent signalling of PKA phosphorylation (Qiu et al. [103]). Other studies 12/13 the P2Y receptor and contribute to rapid actin indicated that regulation of ERK by cAMP involves polymerization and shape changes (Paul et al. [98]). multiple cell type specific mechanisms. In COS cells Signalling towards Rho GTPases is also important in overexpressing "-AR1 or "-AR2, stimulation of these other systems. In brain, ATP and ADP induce receptors activate AC through a G protein-dependent membrane ruffling and chemotaxis of microglial cells mechanism as expected. However, PKA also phos- through G protein-dependent activation of Rac upon phorylates these receptors and induces a switch from stimulation of P2Y receptors (Sasaki et al. [51]; G to G protein binding to "-AR resulting in 12/13 s i/o Purinergic Signalling (2006) 2:451–469 457 activation of ERK upon receptor stimulation (Martin thrombin-induced MEK and ERK activation indepen- et al. [104]). Modulation of the ERK cascade by cAMP dently of Ras or Raf (Nadal-Wollbold et al. [119]). can also occur independently of PKA. In this context, G protein-coupled receptors that are not linked to i/o cAMP binds Epac1 or Epac2, Bexchange protein PLC activation can also modulate mitogenic signalling directly activated by cAMP,’’ GEFs that activate through G"+-dependent activation of PI 3-K+. Signal- Rap1 and Rap2 (de Rooij et al. [105, 106]; Kawasaki ling from PI 3-K+ to ERK proceeds through Shc/Grb2/ et al. [107]). Several examples of G protein-mediated SOS/Ras (Lopez-Ilasaca et al. [86]). An increasing activation of ERK through Epac are reported number of reports point to the involvement of PKCK in (Laroche-Joubert et al. [108]; Lin et al. [109]). Another G protein-dependent phosphorylation of ERK. The PKA-independent mechanism of ERK phosphoryla- first observation was made in CHO cells where tion is the G "+/Src-mediated activation of Ras stimulation of the LPA receptor triggers MEK/ERK (Schmitt and Stork [110]). signalling via a PI 3-K+-dependent activation of PKCK G and some G protein-coupled receptors activate not abrogated by transfection with dominant negative q i/o PLC" and trigger formation of IP and DAG, resulting Ras (Takeda et al. [120]). In addition, a recent report 2+ 2+ in Ca release and PKC activation, respectively. Ca indicated that angiotensin II-induced ERK activation increase can activate ERK through Pyk2 that activates in rat vascular smooth muscle cells requires interaction 2+ Ras as mentioned above. Otherwise, Ca -dependent between Ras and PKCK (Zhao et al. [121]). PKCK- modulation of Ras activity is also mediated by Ras dependent activation of ERK is mediated by inter- guanine nucleotide-releasing factor (RasGRF), a GEF action with MEK, a property shared by other PKC 2+ that contains Ca - and DAG-binding domains, isoforms (Scho ¨ nwasser et al. [122]), or by regulation of 2+ (Ebinu et al. [111]) or by Ca /calmodulin-dependent Raf1. Studies performed in rat embryonic hippocampal kinases CaMK-II and CaMK-IV (reviewed in Agell cells indicated that PKCK can phosphorylate the Raf et al. [112]and Walker et al.[90]). Increase of kinase inhibitory protein (RKIP) resulting in dissocia- intracellular calcium and DAG formation also results tion of the Raf1/RKIP complex (Corbit et al. [123]). In in activation of cPKCs while DAG formation alone is addition, co-immunoprecipitation experiments in COS sufficient to activate nPKCs. Increase in PKC activity cells showed that modulation of c-Raf1 by PKCK is modulates the ERK cascade through Ras by inhibition also regulated by 14-3-3 scaffolding proteins (Van Der of RasGAPs and/or stimulation of RasGEFs. In Hoeven et al. [124]). addition, PKC can activate Raf independently of Ras. Initial studies concerning P2Y receptor-mediated Indeed, it is shown that PKC! phosphorylates Raf at activation of ERK were made in astrocytes where this Ser499 (Kolch et al. [113]). However, mutation of this cascade was shown to be involved in cell proliferation serine residue into alanine does not affect Raf activity and process elongation (Neary and Zhu [125]; King in response to phorbol esters (Yip-Schneider et al. et al. [126]). [114]). More convincing data were obtained when Although ATP triggers pertussis toxin insensitive 2+ constitutively active PKC was expressed in rat 6 IP and Ca responses in astrocytes, these are not fibroblasts. These cells display Ras-independent sig- required for the signalling towards ERK which nalling towards ERK by direct phosphorylation of Raf depends on rapid membrane translocation of PKC% by PKC(. Since activation of Ras is required in several upon phosphatidylcholine hydrolysis by PLD (Neary systems this interaction is cell type-dependent (Cacace et al. [73]). In PC12 cells, stimulation of the P2Y re- et al. [115]; Ueffing et al. [116]). Direct phosphoryla- ceptor also induces PKC%-dependent ERK phosphor- 2+ tion of Raf by PKC is also involved in ERK activation ylation, although this mechanism requires Ca and by the G protein-coupled leukotriene (LT)D receptor Pyk2 for the association of Shc and Grb2 to the i 4 in intestinal epithelial cells. Although stimulation of receptor and for subsequent activation of SOS/Ras/ this receptor also triggers a parallel PKC-independent Raf/MEK/ERK (Soltoff et al. [84]). Many reports activation of Ras, transfection experiments confirmed showed that P2Y receptor-mediated ERK signalling that Ras is dispensable for LTD receptor-mediated requires PKC activation (Graham et al. [127]; Huwiler ERK activation (Paruchuri et al. [117]). When consti- and Pfeilschifter [128]; Erlinge [129]), but a PKC- tutive active point mutants of PKC!, PKC% and PKC( independent mechanism is reported in thyroid FRTL- were introduced in COS cells, only PKC% activated the 5 cells (Tornquist et al. [130]). In 1321N1 astrocytoma ERK cascade (Ueda et al. [118]), indicating that cells, stimulation of the P2Y receptor with UDP acti- involvement of PKC isoforms in ERK signalling vary vates PKC!, ( and K which are correlated with ERK among different cell types. This is also confirmed by the phosphorylation (Kim et al. [131]). Although the use of observation that, in platelets, cPKCs are involved in general PKC inhibitors 3-[1-(dimethylaminopropyl) 458 Purinergic Signalling (2006) 2:451–469 indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride dependent activation of p38 and ERK1/2 (Berenbaum (GF109203X) and 12-(2-cyanoethyl)-6,7,12,13-tetrahy- et al. [136]). In primary astrocytes, P2Y receptor- dro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)- mediated ERK activation by ATP is shown to induce carbazole (Go ¨ 6976) diminished ERK signalling, the reactive astrogliosis, a phenomenon that occurs upon lack of specificity of these compounds makes it dif- brain injury and is characterised by astroglial prolifer- ficult to determine the contribution of each of these ation, cellular hypertrophy and up regulation of GFAP. PKC isoforms in the mechanism of ERK activation This effect is mediated by an ERK-dependent increase (Way et al. [132]). in the expression of COX-2 (Brambilla et al. [53]). In Enhanced proliferation by a P2Y receptor-mediated C6 cells, P2Y and P2Y receptor-induced activation 12 2 stimulation of the ERK pathway has been reported in of ERK is coupled to an enhanced cell proliferation, a large number of cell types such as human mesangial while a negative modulation of GFAP synthesis by the cells, vascular smooth muscle cells and primary astro- P2Y receptor is reported (Claes et al. [39]; Tu et al. cytes (Huwiler and Pfeilschifter [128]; Harper et al. [44]; Van Kolen and Slegers [45]). These differences [133]). Transient ERK activation by P2Y , P2Y or are probably due to the fact that in C6 cells induction 1 2 P2Y receptor stimulation also increases cell prolifer- of GFAP expression is not correlated with an en- ation in C6 cells indicating that activation of several hanced proliferation but requires growth arrest. P2Y receptor subtypes can converge into the same In summary, most P2Y receptors are coupled to physiological response (Table 2) (Tu et al. [44]; Claes ERK phosphorylation, but the signalling mechanism et al. [39]; Czajkowski et al. [46]). and the physiological effect of this pathway are cell type In addition to mitogenesis, P2Y receptor signalling specific and are determined by the cellular context. towards ERK elicits other physiological processes including cell survival, inflammation and reactive PI 3-K/PKB signalling gliosis. In human lung microvascular endothelial cells, hyperoxia-induced release of ATP results in cell PKB/Akt is involved in a large variety of cellular survival through ERK and PI 3-K signalling cascades processes including glucose metabolism, mitogenesis, activated by P2Y and/or P2Y receptors, while 2 6 stimulation of the ERK cascade by the P2Y receptor differentiation, survival and motility (Brazil et al. [137]). This member of the AGC protein kinase protects 1321N1 astrocytoma cells from TNF!-induced apoptosis (Ahmad et al. [134]; Kim et al. [131]). Rapid superfamily is recruited to the plasmamembrane upon PI 3-K-mediated PIP3 formation, but is also controlled ERK1/2 and p38 MAPK activation plays an important role in P2Y receptor-dependent primary granule in a PI 3-K-independent, but calmodulin-dependent, 2+ fashion upon intracellular Ca mobilisation by stimu- release from human neutrophils (Meshki et al. [135]). A similar phenomenon is observed in articular chon- lation of neuronal NMDA receptors (Cantley [138]; Leevers et al. [139]; Woodgett [140]; Yano et al. [141]). drocytes where ATP acts as a pro-inflammatory medi- ator by increasing arachidonic acid production and Modulation of PKB activity is reported for a variety release of prostaglandin E through a P2Y receptor- of GPCR ligands including adrenergics, cannabinoids, 2 2 Table 2 G protein-dependent modulation of ERK and PKB signalling cascades in C6 cells. G protein ERK PI 3-K/PKB Effect 2+ P2Y G j PLC-PKC-Ca -Ras. , Attenuation of PI 3-K Proliferation 1 q activated by growth factors 2+ P2Y G j PLC-PKC-Ca -Ras. – Proliferation 2 q P2Y G j RhoA-PKC-Raf-MEK j PI 3-K/PKB Proliferation/inhibition of 12 i astrocytic differentiation 2OR G j FGF transactivation – Proliferation "-AR G , Transient inhibition , Transient inhibition Growth arrest/astrocytic dependent on cAMP by cAMP differentiation CB G , Sustained inhibition , Sustained inhibition Growth arrest/apoptosis Transient ERK activation by P2Y (Czajkowski et al. [46]), P2Y inhibits ERK and PKB concomitant with induction of differenti- 1 2 (Tu et al. [44]), P2Y (Grobben et al. [40]) and 2 opioid receptors ation (Wang et al. [149]; Van Kolen and Slegers [45]). Inhibition of (2OR) (Belcheva et al. [198]) enhances cell proliferation while these pathways by cannabinoids (CB) is sustained and induces stimulation of the "-adrenergic receptor ("-AR) transiently apoptosis (Ellert- Miklaszewska et al. [184]). Purinergic Signalling (2006) 2:451–469 459 carbachol, glutamate, histamine, nucleotides and ATP or UTP activates PKB by a PDK-1-dependent thrombin (Dickenson [142]; Franke et al. [143], mechanism while, in C6 cells, ADP activates PI 3-K/ Iacovelli et al. [144] Murga et al. [145]; Sanchez et al. PKB by the G protein-coupled P2Y receptor but i 12 [146]). Due to the existence of multiple phosphoinosi- inhibits PI 3-K by stimulation of the G /G protein- q 11/12 tide-dependent cascades, regulation of PKB signalling coupled P2Y receptor (Table 2) (Van Kolen and by GPCRs varies among the studied systems. Slegers [45]; Czajkowski et al. [46]; Huwiler et al. In HEK293 cells, stimulation of "-AR with [159]). Although most effects of P2Y-mediated activa- (-)-isoproterenol activates PKB via G "+, Src, Ras and tion of PI 3-K signalling are known to be related to cell PI 3-K (Schmitt and Stork [110]; Bommakanti et al. proliferation, differentiation and survival, this signal- [147]) while activation of AC by G! exerts differential ling cascade is also involved in other processes. In this effects on PKB activity. In cells expressing Epac, cAMP regard, it can be mentioned that P2Y receptor- activates PI 3-K/PKB via Rap1 while, in other cells, mediated PI 3-K/PKB activation modulates prolifera- cAMP activates PKA that exerts a negative action on PI tion and differentiation of C6 cells, but also plays an 3-K and PKB (Mei et al. [148]; Wang et al. [149]). important role in ADP-induced platelet aggregation G protein-mediated activation of PKB can occur (Van Kolen and Slegers [45]; Czajkowski et al. [46]; through the coupling of the G"+ subunit to the catalytic Chen et al. [160]; Kim et al. [161]). subunit of PI 3-K or via growth factor receptor trans- activation. Although only p110+ was initially reported to be activated by G"+ subunits, this feature is also P2Y receptor-integrated G protein-coupled receptor observed for the p110" isoform (Kurosu et al. [150]; and receptor tyrosine kinase signalling cascades Stoyanov et al. [151]). This mechanism is reported in Vero cells where stimulation with LPA activates Ras G protein-coupled receptor cross-talk upon increase in p110" lipid kinase activity (Yart et al. [152]). G protein-mediated transactivation of Complementary to vertical downstream signalling upon growth factor receptors is reported in HaCaT, A-431, GPCR stimulation, these receptors also mediate lateral and HEK293 cells where stimulation of the angioten- signalling by cross-talk with other receptors (reviewed in sin type I receptor by mechanical stress induces Cordeaux and Hill [162]). In human platelets, it was transactivation of EGFR leading to activation of the reported that P2Y receptor activation potentiates 2+ PI 3-K/PKB cascade and protection of these cells from P2Y receptor-mediated Ca signalling, while the apoptosis (Kippenberger et al. [153]). P2Y receptor negatively regulates this action (Hardy In 1321N1 astrocytoma cells, PLC" activation by the et al. [163]). In renal mesangial cells, P2Y receptors G protein-coupled muscarinic M receptor also trig- activated by ATP and UTP induce a rapid desensitisa- q 3 gers PI 3-K activation through ErbB3 transactivation, tion of the sphingosine-1-phosphate (S1P) receptor by 2+ but this mechanism requires Ca mobilisation (Tang PKC-dependent phosphorylation (Xin et al. [164]). A et al. [154]). In contrast, some reports showed an more complex interplay is observed between P2Y inhibitory pathway from G protein-coupled receptors receptors and 5-HT receptor subtypes. Studies per- towards PI 3-K by direct interaction between G!- formed in CHO cells stably expressing 5-HT recep- 1A subunits released from heterotrimeric G proteins and tors revealed that the responsiveness of this receptor is p110!, as reported for the ! -AR in rat-1 fibroblasts reduced by a PLD/PKC-dependent phosphorylation 1A (Ballou et al. [155, 156]), or by inhibition of insulin upon short (<5 min) pre-treatment with ATP, while receptor substrate-1-associated PI 3-K activity in the agonist efficacy of the overexpressed 5-HT 1B 1321N1 astrocytoma cells by carbachol, histamine or receptor is not altered. Alternatively, longer treatment thrombin. These observations reveal opposing effects with ATP alone attenuates 5-HT signalling by a 1B of muscarinic receptor stimulation on PI 3-K activity mechanism that requires activation of phospholipase mediated by insulin and ErbB3 receptors in these cells A (PLA ) (Berg et al. [165]). Furthermore, stimulation 2 2 (Batty et al. [157]). of P2Y receptors can also modulate the release of Modulation of PI 3-K/PKB signalling is also reported transmitter molecules, including dopamine, glutamate for a few P2Y receptors. In bovine adventitial fibro- and serotonin (Bezzi and Voltera [52]; Krugel et al. blasts, ATP is shown to induce proliferation through [166]; Nedergaard et al. [167]). A recently discovered parallel but independent ERK and PI 3-K signalling mechanism of GPCR cross-talk is the assembly of a cascades that contribute to mTOR and p70S6K phos- heteromeric receptor complex displaying the pharmaco- phorylation (Gerasimovskaya et al. [158]). In rat logical profile of one receptor and the signalling proper- mesangial cells, stimulation of the P2Y receptor with ties of the other. Such an interaction is reported in 2 460 Purinergic Signalling (2006) 2:451–469 HEK293 cells overexpressing A and P2Y receptors. Pyk2, probably by its proline-rich putative SH3 1 1 The heteromeric A -P2Y receptor complex inhibits binding sites (PXXP). This interaction is implicated 1 1 AC through G protein, but displays P2Y receptor- in P2Y receptor-induced transactivation of EGF, i/o 1 2 like pharmacological properties (Yoshioka et al. [168]). PDGF and VEGF receptors (Liu et al. [176]; Seye et al. [100]). Src inhibition abolishes growth factor P2Y receptor-mediated transactivation receptor transactivation and ERK phosphorylation. Although the rat P2Y receptor lacks PXXP motives, Many studies reveal that GPCRs and growth factor tyrosine kinase-dependent activation of ERK upon receptors share a number of signalling modules P2Y receptor stimulation is reported in a few rat cell (e.g., Raf/MEK/ERK, PI 3-K/PDK/PKB) to transduce lines, including C6 and PC12 cells (Soltoff et al. [84]; their effects. In the past decade, it has become clear Tu et al. [44]). In the latter cases, P2Y receptor- that the signalling pathways of both receptor systems dependent activation of Pyk2 is mediated by PKC and 2+ are interconnected. Stimulation of a GPCR can induce Ca suggesting that the PXXP sequence is dispens- a rapid tyrosine phosphorylation of RTKs. This trans- able for P2Y receptor-induced tyrosine phosphoryla- activation mechanism is reported for many GPCRs tion of Pyk2 and downstream signalling towards ERK. and proceeds through the G"+ subunit-dependent Moreover, P2Y mutants lacking PXXP-motives are activation of Src. Src in turn activates RTKs by still able to activate ERK demonstrating the existence phosphorylation of specific tyrosines located in their of other pathways towards phosphorylation of ERK intracellular domains or induction of matrix metal- (Liu et al. [176]). Observations made in human loproteases-dependent release of growth factor recep- endothelial cells, where UTP-induced signalling to 2+ tor ligands, e.g., release of heparin-bound EGF ERK was shown to depend on Ca ,PKC and (Luttrell and Luttrell [169]). integrin-mediated cell anchorage, already pointed to Another target for signal integration of GPCRs and a pathway distinct from the classical Ras/Raf/MEK/ RTKs are docking proteins. Although these proteins ERK cascade (Short et al. [177]). Human and mouse contain phospho-tyrosine binding domains that inter- P2Y receptors contain a RGD sequence which allows act with phosphorylated tyrosine residues of RTKs, activation of ERK by interaction with ! " /" integrins V 3 5 stimulation of GPCRs can induce growth factor followed by G protein coupling. Since these proteins receptor-independent phosphorylation of docking pro- also mediate cell adhesion and chemotaxis, the ob- teins by Src (Bisotto and Fixman [170]). served P2Y /! " /" -interaction also points to a possi- 2 V 3 5 In addition to GPCR-dependent phosphorylation of ble function of P2Y receptors in inflammatory RTKs, the opposite activation mechanism is also responses (Erb et al. [178]). reported. Binding of PDGF to its cognate receptor It is clear that, in analogy with other GPCRs, cross- induces association of PDGFR with the G protein- talk between P2Y and growth factor receptors may coupled S1P receptor. Subsequently, Src is recruited to occur at different levels of the signal transduction this complex by G"+ subunits and phosphorylates pathway depending on receptor subtypes and on the Grb-2 associated binder-1 resulting in dynamin studied system. For the P2Y receptor, additional II-induced Bpinching off’’ of vesicles involved in transactivation mechanisms are facilitated by the endocytosis of PDGF-S1P signalling complexes and presence of signalling motives (e.g., PXXP or RGD) subsequent activation of ERK1/2 (Waters et al. [171]). that allow direct interaction with other signalling Cross-talk between RTKs and P2Y receptors is components (Src, integrins). reported in Mu ¨ ller glial cells where ATP exerts its mitogenic effect through transactivation of EGF and PDGF receptors resulting in ERK-dependent en- P2Y receptor-activated signal transduction pathways hanced proliferation. In these cells, ATP-induced in C6 glioma cells activation of ERK was abolished by treatment with the RTK autophosphorylation inhibitor tyrphostin As mentioned above, the final outcome of nucleotide- (AG1478) (Milenkovic et al. [172]). In rat striatal mediated signalling is influenced by ecto-enzymes astrocytes, ATP and bFGF activate ERK and induce (Claes and Slegers [17]; Czajkowski and Baranska astrogliosis by a mechanism that is insensitive to [18]; Grobben et al. [21, 179]). ATP and ADP hy- RTK inhibition (Abbracchio et al. [173]; Bolego et al. drolysis to adenosine results in growth inhibition by a [174]; Neary et al. [175]). More recently, mechanistic mechanism that is not yet fully understood. When studies performed in 1321N1 astrocytoma cells reveal nucleotide hydrolysis is prevented, ATP, ADP and that the human P2Y receptor interacts with Src and Ap A (in particular Ap A and Ap A) increase cell 2 n 3 4 Purinergic Signalling (2006) 2:451–469 461 2+ proliferation more than two-fold. Stimulation with activates ERK through a Ca -dependent mechanism 2MeSADP, a P2Y agonist not hydrolysed by the (Czajkowski et al. [46]), likely by a similar mechanism ecto-enzymes present on the plasma membrane of C6 as reported for the P2Y receptor (Tu et al. [44]). In cells, also results in growth enhancement and inhibi- addition, it has been shown that ADP can activate tion of "-AR-induced differentiation into astrocyte ERK by stimulation of the P2Y receptor through a type II (Claes et al. [39]; Van Kolen and Slegers [45]). RhoA- and PKC-dependent pathway that does not 2+ The pathways involved in the P2Y receptor-dependent require Ca , Ras or tyrosine kinase activation 2+ effects on growth and differentiation of these cells are (Grobben et al. [40]). The fact that Ca removal does presented in Figure 1. not affect P2Y receptor-mediated ERK activation Nucleotides stimulate several purinergic receptors excludes the involvement of cPKCs. Stimulation of the that activate the ERK cascade by at least two distinct P2Y receptor does not induce PI-turnover, but mechanisms. The P2Y receptor, stimulated by UTP nPKCs might be involved since alternative activation and ATP, enhances ERK phosphorylation through a mechanisms, based on Ser/Thr and Tyr phosphoryla- PLC"/PKC/Ras/Raf/MEK cascade that is attenuated tion, have been reported (Steinberg [180]; Parekh et al. 2+ by inhibition of tyrosine kinases and Ca chelation by [181]). Data from our laboratory suggest an important 2+ BAPTA-AM (Tu et al. [44]). The Ca -dependence of role for PKCK in P2Y receptor-dependent activation the P2Y receptor-mediated activation of ERK sug- of ERK. The fact that no cross-talk between ERK and gests the involvement of a cPKC (!, "I, "II or +). It is PI 3-K is observed in C6 cells indicates that PKCK also shown that ADP stimulates the P2Y receptor and exerts its actions independently of PI 3-K via a RhoA- Figure 1 Overview of P2Y receptor-mediated signalling cascades in C6 cells. Green and red lines represent stimulatory (green arrows) Figure 1 Overview of P2Y receptor-mediated signalling cascades lation also inhibits cAMP-dependent induction of differentia- and inhibitory (red squares) actions respectively. Dashed lines are incomplete characterised pathways. P2Y receptor stimulation in C6 cells. Green and red lines represent stimulatory (green tion by reactivation of PKB which requires Src/Pyk2 complex enhances ERK-dependent proliferation through a PLC-dependent pathway while P2Y receptor stimulation enhances cell arrows) and inhibitory (red squares) actions respectively. Dashed formation and Rap1 activation. Formation of the Src/Pyk2 proliferation by RhoA- and PKCK-dependent activation of ERK (Claes et al. [39]; Grobben et al. [40]; Tu et al. [44]; Van Kolen 2+ lines are incomplete characterised pathways. P2Y receptor complex requires Ca and PLD2 which is constitutively active and Slegers, unpublished data). P2Y receptor stimulation also inhibits cAMP-dependent induction of differentiation by reactivation stimulation enhances ERK-dependent proliferation through a (Claes et al. [22]; Van Kolen and Slegers [45]; Van Kolen et al. 2+ of PKB which requires Src/Pyk2 complex formation and Rap1 activation. Formation of the Src/Pyk2 complex requires Ca and PLD2 PLC-dependent pathway while P2Y receptor stimulation [185]). Cyclic AMP-dependent inhibition of PKB and ERK is which is constitutively active (Claes et al. [22]; Van Kolen and Slegers [45]; Van Kolen et al. [185]). Cyclic AMP-dependent inhibition enhances cell proliferation by RhoA- and PKCK-dependent suggested to depend on inhibition of Rap1 (Wang et al. [149]). of PKB and ERK is suggested to depend on inhibition of Rap1 (Wang et al. [149]). The negative modulation of PI 3-K by the P2Y activation of ERK (Claes et al. [39]; Grobben et al. [40]; Tu The negative modulation of PI 3-K by the P2Y receptor is only et al. [44]; Van Kolen and Slegers, [199]). P2Y receptor stimu- displayed in the presence of serum (Czajkowski et al. [46]) 12 462 Purinergic Signalling (2006) 2:451–469 dependent mechanism (Grobben et al. [40]; Van Kolen inhibition of PKB while recovery of this activity is and Slegers, [199]). Although P2Y receptors use required to prohibit cell death. This hypothesis is different mechanisms to activate ERK, they all con- confirmed by a recent study where sustained inhibition verge to increased cell proliferation by enhanced of PI 3-K/PKB by cannabinoids is shown to induce synthesis of c-Myc, c-Jun and c-Fos (Zhang et al. apoptosis in C6 cells (Table 2) (Ellert-Miklaszewska [182]). Progression through the G1/S phase of the cell et al. [184]). Taken together, P2Y receptor stimula- Kip cycle is due to a decreased expression of p27 and tion inhibits cAMP-dependent induction of differenti- increased expression of cyclinD. ation by a transient increase in PI 3-K/PKB activity. 2+ While stimulation of ERK signalling by P2Y recep- Ca chelation inhibits the basal PKB activity and tors has been known for several years, the coupling with P2Y receptor-mediated increase in PKB phosphoryla- PI 3-K activation was discovered more recently. When tion. Although C6 cells also express the P2Y receptor, C6 cells are grown in the presence of serum, P2Y stimulation with UTP does not enhance the activity of receptor signalling predominates and is shown to PI 3-K/PKB, which may be explained by a differential inhibit PI 3-K (Czajkowski et al. [46]). Upon serum coupling to G protein subtypes. P2Y receptor-mediat- deprivation, P2Y receptor expression decreases while ed signalling proceeds through G proteins while the 1 q P2Y becomes the main ADP-stimulated receptor activation of PDK is G protein-dependent (Table 2) 12 i that enhances the activity of PI 3-K by a G protein- (Tu et al. [44]; Huwiler et al. [159]). The lack of G i i dependent mechanism. These observations demon- protein coupling of the P2Y receptor in C6 cells might strate that, in addition to autocrine growth factor be a consequence of compartimentalisation into cav- receptor signalling, the constitutive PI 3-K activity in eolae as reported for some G protein-coupled recep- C6 cells is modulated by P2Y and P2Y receptor tors (Bhatnagar et al. [81]). 1 12 expression. Another cross-talk at the level of PI 3-K/ Although experiments in CHO cells reveal that P2Y PKB is observed for P2Y and "-AR. Increase in receptor-induced ERK activation requires PI 3-K+ cAMP upon stimulation of the latter receptor tran- (Souletetal. [92]), experiments performed with siently inhibits PKB phosphorylation. Stimulation of LY294002 or Wortmannin excluded cross-talk between the P2Y receptor, which negatively affects AC, does both cascades in C6 cells (Grobben et al. [40]). These not only counteract this inhibition but even enhances differences in signalling mechanisms can be explained PKB activity in comparison to unstimulated cells, by the fact that the latter PI 3-K-isoform is only suggesting that P2Y receptor-mediated PI 3-K/PKB moderately expressed in C6 cells (Van Kolen and activation is not only due to its inhibitory effect on AC Slegers [45]). The exact mechanism of P2Y receptor- (Van Kolen and Slegers [45]; Czajkowski et al. [46]; induced PI 3-K/PKB activation is not fully understood, Baranska et al. [183]). In addition to their opposing but recent data revealed that Src and Pyk2 are involved effects on PI 3-K/PKB signalling, unpublished data of in P2Y receptor signalling to PI 3-K (Van Kolen et al., our laboratory revealed similar modulation of ERK [185]). A similar pathway is observed in PC12 cells signalling by P2Y and "-AR. Whether the P2Y where Src, in complex with Pyk2 and PLD2, activates 12 12 receptor-mediated reversal of ERK inhibition is in- PI 3-K in response to H O (Banno et al., [186]). Since 2 2 volved in the inhibition of "-AR-induced GFAP PLD2 is constitutively active in C6 cells (Bobeszko synthesis remains to be determined. The observation et al. [187]), a significant role for this enzyme in PI 3-K/ that stimulation of the cells with UTP activates ERK, Akt signalling is suggested. Although Soulet et al. [92] but fails to inhibit the "-AR-induced growth arrest and reported that transactivation of PDGFR is involved in GFAP synthesis, suggests that ERK activation alone is PI 3-K activation by the P2Y receptor in CHO cells, not sufficient to counteract differentiation (Claes et al. the use of receptor kinase inhibitors indicated that [39]; Tu et al. [44]). Conversely, transfection of C6 cells PDGFR and EGFR are not transactivated by the P2Y with constutively active PKB prevented (-)-isoproter- receptor in C6 cells. Alternatively, a Rap1-mediated enol-induced differentiation indicating that inhibition activation of PI 3-K by the P2Y receptor cannot be of PKB signalling is required for cAMP-dependent excluded. Indeed, PI 3-K is postulated as a downstream induction of differentiation. Apparently this observa- effector of Rap1 that is inhibited by an increase in tion is in contrast with data showing that cAMP- cAMP concentration (Wang et al. [149]). Data from our dependent induction of differentiation requires PI 3-K laboratory indicated a rapid P2Y receptor-induced 2+ activity which is not inhibited upon a 48-h treatment activation of Rap1 that was abolished by Ca chelation with dbcAMP (Roymans et al. [34]). This might be and inhibition of Src/Pyk2 complex formation but not explained by the fact that induction of differentiation by PI 3-K inhibition (Van Kolen et al. [185]). These by stimulation of "-AR proceeds through transient results positioned Rap1 downstream of Src/Pyk2 but Purinergic Signalling (2006) 2:451–469 463 upstream of PI 3-K. In addition, this mechanism determined by differential expression of signalling 2+ involves G"+ protein subunits and Ca -dependent proteins, but on the other hand also depends on the activation of Pyk2 that requires association to IGF-IR assembly of signalling modules. Besides specific pro- and PLD2 to interact with Src. Although Src and Pyk2 tein-protein interactions, intracellular compartmentali- are shown to activate Ras/Raf/MEK/ERK in primary sation (e.g., lipid rafts, clathrin-coated vesicles) also astrocytes (Wang and Reiser [83]), this mechanism did contributes to the specificity of receptor signalling. not contribute to P2Y receptor-mediated ERK acti- Identification of the signalling modules and cellular vation in C6 cells pointing to a physical separation of compartmentalisation will provide more insight into the both cascades (Grobben et al. [40]; Van Kolen and P2Y receptor-activated signalling cascades. Slegers, [199]). Indeed, the formation of a Pyk2/Src/ PLD2/IGFI-R complex may contribute to compartmen- Acknowledgment This work was supported by grants from the talisation of this signalling pathway that requires intact Fund for Scientific Research Flanders (HS) and BOF-NOI (HS). lipid rafts to be active (Van Kolen et al. [185]). 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Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks

Purinergic Signalling , Volume 2 (3) – Jun 7, 2006

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Abstract

Purinergic Signalling (2006) 2:451–469 DOI 10.1007/s11302-006-9008-0 Integration of P2Y receptor-activated signal transduction pathways in G protein-dependent signalling networks Kristof Van Kolen & Herman Slegers Received: 19 July 2005 / Accepted: 17 March 2006 / Published online: 7 June 2006 # Springer Science + Business Media B.V. 2006 Abstract The role of nucleotides in intracellular energy Abbreviations provision and nucleic acid synthesis has been known for AC adenylate cyclase 1 3 a long time. In the past decade, evidence has been ApAP ,P -di(adenosine-5 )triphosphate 1 4 presented that, in addition to these functions, nucleo- ApAP ,P -di(adenosine-5 )tetraphosphate tides are also autocrine and paracrine messenger AR adrenergic receptor molecules that initiate and regulate a large number of COX cyclooxygenase biological processes. The actions of extracellular nucle- DAG diacylglycerol otides are mediated by ionotropic P2X and metabo- ERK extracellular signal-regulated kinase tropic P2Y receptors, while hydrolysis by ecto-enzymes GFAP glial fibrillary acidic protein modulates the initial signal. An increasing number of GPCR G protein-coupled receptor studies have been performed to obtain information on HT hydroxytryptamine the signal transduction pathways activated by nucleo- IP inositol (1,4,5)-triphosphate 0 0 tide receptors. The development of specific and stable PAP adenosine-3 ,5 -biphosphate purinergic receptor agonists and antagonists with ther- PI phosphatidylinositol apeutical potential largely contributed to the identifi- PL phospholipase cation of receptors responsible for nucleotide-activated PI 3-K phosphatidylinositol 3-kinase pathways. This article reviews the signal transduction PPADS pyridoxalphosphate-6-azophenyl-2 , pathways activated by P2Y receptors, the involved 4 -disulphonate second messenger systems, GTPases and protein Pyk2 proline-rich tyrosine kinase 2 kinases, as well as recent findings concerning P2Y RKIP Raf kinase inhibitory protein receptor signalling in C6 glioma cells. Besides vertical RTK receptor tyrosine kinase signal transduction, lateral cross-talks with pathways activated by other G protein-coupled receptors and growth factor receptors are discussed. Introduction . . . . Key words C6 glioma ERK P2Y receptors PKB Pharmacological properties of P2Y receptors transactivation tyrosine kinases Extracellular actions of adenine nucleotides were ini- tially characterised in the cardiovascular system by Drury and Szent-Gyorgyi [1]. It took more than four K. Van Kolen H. Slegers (*) Department of Biomedical Sciences, Cellular Biochemistry, decades before the concept of purinergic signalling was University of Antwerp, accepted, but now it is well established that nucleo- Universiteitsplein 1, tides initiate and regulate a variety of biological 2610 Wilrijk-Antwerpen, Belgium processes, including neurotransmission, inflammation, e-mail: herman.slegers@ua.ac.be regulation of blood pressure, platelet aggregation, cell K. Van Kolen growth and differentiation (Abbracchio et al. [2]; CNS research, Johnson & Johnson, PRD, Burnstock and Williams [3]; Burnstock [4]; Ralevic Janssen Pharmaceutica, and Burnstock [5]). Beerse, Belgium 452 Purinergic Signalling (2006) 2:451–469 Nucleotides are released in the extracellular fluid by tides (P2Y ). The pharmacological profile of the cell lysis, exocytosis, secretion of granules, efflux and recently cloned P2Y receptor is distinct from the upon cellular stress such as changes in osmolarity and other P2Y receptors since UDP-glucose, UDP-galac- mechanical perturbations. Once released, they mediate tose, UDP-glucuronic acid and UDP-N-acetylglucos- their effect by stimulation of nucleotide receptors. amine are specific ligands of this receptor (Chambers Based on pharmacological properties, the first sug- et al. [13]). Natural P2Y receptor ligands do not gestion for the existence of ionotropic P2X receptors exclusively bind to one receptor subtype. ADP is an and metabotropic P2Y receptors was made by agonist of P2Y ,P2Y and P2Y receptors, whereas 1 12 13 Burnstock and Kennedy [6]. After cloning, multiple ATP is a full agonist of P2Y and P2Y ,but a partial 2 11 subtypes of P2X and P2Y receptors were characterised agonist or antagonist of P2Y ,P2Y and P2Y 1 12 13 unambiguously (Abbracchio and Burnstock [7] receptors. Although the pharmacological properties of Burnstock and Williams [3]; Fredholm et al. [8]). P2Y receptors (Table 1) are well conserved between Up to now, the P2Y receptor family comprises at least species, some remarkable differences have been ob- eight subtypes, P2Y ,P2Y ,P2Y ,P2Y ,P2Y ,P2Y , served. While UTP acts as an agonist of both human and 1 2 4 6 11 12 P2Y and the recently identified P2Y receptor rat P2Y receptors, ATP is a potent agonist of the rat 13 14 4 (Ralevic and Burnstock, [5] Abbracchio et al. [9]; P2Y , but an antagonist of the human orthologue. Communi et al. [10]; Hollopeter et al. [11]; Zhang Mutational analysis revealed that the second extracel- et al. [12]). According to the agonist profile, P2Y lular loop of the P2Y receptor is responsible for the receptors can be subdivided into receptors responding opposing effect of ATP in both species (Herold et al. to adenine mono- and dinucleotides (P2Y , P2Y , [14]). A similar phenomenon is observed when human 1 11 P2Y ,P2Y ), and to uridine nucleotides (P2Y , and canine P2Y receptors were stably expressed in 12 13 4 11 P2Y ), and receptors for adenine and uridine nucleo- CHO-K1 and 1321N1 astrocytoma cells. Whereas the Table 1 Pharmacological profile of P2Y receptors and second messenger systems. Agonists Antagonists Effector G protein P2Y 2MeSADP, ADP, ADP"S, Suramin, PPADS, PAP, MRS2179, MRS2216, PLC, I G /G 1 K, Ca q 11/12 Ap A, MRS2365 MRS2279, MRS2500, MRS 2603 P2Y UTP+S, ATP+S, UTP, ATP, Suramin PLC, I G /G 2 K, Ca i q INS37217, Ap A a a P2Y UTP+S, UTP, ATP ATP , PPADS PLC, I G /G 4 K q 11/12 P2Y UDP"S, UDP, UTP, INS48823 PPADS, suramin, MRS2567 PLC, I G /G 6 K q 11/12 P2Y ATP!S, ATP+S, ATP Suramin AC, PLC G /G 11 q s P2Y 2MeSADP, ADP, Ap A, ATP, AR-C69931MX, AR-C67085, AR-C78511KF, AC, I G 12 3 K, Ca i/o Ap A clopidogrel, 2MeSAMP, DIDS, suramin, MRS2395 P2Y 2MeSADP, ADP, Ap A, ATP AR-C69931MX, Ap A, PPADS, suramin, MRS2211, AC, PLC, I G /G 13 3 4 Ca i q MRS2603 P2Y UDP-glucose, UDP-galactose, AC, I G 14 Ca i UDP-glucuronic acid, UDP-N-acetylglucosamine ATP acts as an agonist of the rat P2Y but as an antagonist of acid monoammonium salt; DIDS, 4,4 -diisothiocyanatostilbene-2, 0 0 0 the human P2Y receptor (Herold et al [14]). Reactive blue 2 is not 2 -disulphonic acid; INS37217 [P(1)-(uridine 5 )-P (4)-(2 -deoxy- included in the list since it displays lack of specificity towards the cytidine 5 )tetraphosphate tetrasodium salt; INS48823 P -((2-benzyl-1, 3 6 0 0 different P2Y subtypes. References: Abbracchio et al. [9]; Com- 3-dioxolo-4-yl)uridine 5 )P -(uridine 5 ) triphosphate; MRS2179, N - 0 0 0 0 muni et al. [10, 191]; Chambers et al. [13]; Claes and Slegers [17]; methyl-2 -deoxyadenosine-3 ,5 -bisphosphate; MRS2211, pyridoxal- 5 - Kim et al. [26]; Xu et al. [27]; Boyer et al. [38, 189, 190]; Grobben phosphate-6-azo-(2-chloro-5-nitrophenyl)-2,4-disulphonate; MRS2216, 0 0 0 et al. [40]; Marteau et al. [47]; Filippov et al. [57– 60, 63]; Simon 2 -deoxy-2-chloro-N -methyladenosine-3 ,5 -bisphosphate; MRS2279, 0 0 0 et al. [61]; Wirkner et al. [62]; Korcok et al. [192]; Muller [193]; 2-chloro-N -methyl-(N)-methanocarba-2 -deoxyadenosine 3 ,5 -bis- 0 0 0 0 0 Skelton et al. [194]; Yerxa et al. [195]; Jacobson et al. [196]; von phosphate; MRS2365, [(1 S,2 R,3 S,4 R,5 S)-4-[(6-amino-2-methylthio- 1 3 Ku¨ gelgen [197]. Abbreviations: Ap A, P ,P -di(adenosine-5 ) 9H-purin-9-yl)-1-diphosphoryloxymethyl]bicyclo[3.1.0]hexane-2, 3-diol]; 1 4 triphosphate; Ap A, P ,P -di(adenosine-5 )tetraphosphate; AR- MRS2395, 2-dimethyl-propionic acid-3-(2-chloro-6-methylaminopurin- C69931MX, N -(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio) 9-yl)-2-(2,2-dimethylpropionyloxy-methyl)-propylester; MRS2500, 2-io- 0 0 0 -",+-dichloromethylene ATP; AR-C67085, 2-propylthio-D-"+- do-N -methyl-(N)-methanocarba-2 -deoxyadenosine 3 ,5 -bisphosphate; dichloromethylene adenosine 5 -triphosphate; AR-C78511KF, MRS2567, 1,2-di-(4-isothiocyanatophenyl)ethane; MRS2603, pyridox- (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyri- al-5 -phosphate-6-azo-(4-chloro-3-nitrophenyl)-2,4-disulphonate; 0 0 midin-3-yl]-1,5,6-trideoxy-"-D-ribo-hept-5-enofuranuronoyl]-L-aspartic PPADS, pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonic acid. Purinergic Signalling (2006) 2:451–469 453 human P2Y is potently activated by adenosine tri- cAMP (dBcAMP) or 8-chloro-cAMP, induces differ- phosphate nucleotides, the canine orthologue displayed entiation into an astrocyte type II (Roymans et al. more selectivity towards the corresponding diphos- [34]). During this process, cessation of cell growth is phates. In this case, the nucleotide selectivity is due to accompanied by a shift in intermediate filament differences in the amino acid sequence at the juxtapo- synthesis from vimentin to glial fibrillary acidic protein sition of TM6 and the third extracellular loop also (GFAP) (Backhovens et al. [35]). The latter protein is reported to play an important role in agonist selectivity an astrocytic differentiation marker whose expression and signalling of other G protein-coupled receptors is regulated by cAMP at the transcriptional and (GPCR) (Qi et al. [15]; Lawson and Wheatley [16]). translational level (Messens and Slegers [36]). Despite their chemical stability, extracellular nu- In our laboratory, the signalling pathways activated by cleotides are metabolised by several ecto-enzymes extracellular nucleotides, and in particular those affecting (Claes and Slegers [17]; Czajkowski and Baranska cell proliferation and differentiation of C6 cells, were [18]; Goding et al. [19]; Zimmerman [20]). Extracellu- studied in detail. The presence of a P2Y receptor on these lar hydrolysis complicates the evaluation of nucleo- cells that negatively affects adenylate cyclase (AC) was tide-mediated effects on different cell types and can be postulated for more than a decade (Pianet et al. [37]; Boyer overcome by the use of specific non-hydrolysable et al. [38]). This receptor is coupled to a G protein and receptor agonists or ecto-enzyme inhibitors. Some has been denominated P2Y - (Claes et al. [39]; Grobben AC P2Y receptor antagonists, such as pyridoxalphosphate- et al. [40]) before its identification as the P2Y receptor 0 0 6-azophenyl-2 ,4 -disulfonic acid (PPADS), suramin, initially cloned from blood platelets (Czajkowski et al. 0 0 reactive blue 2 and 4,4 -diisothiocyanatostilbene-2,2 - [41]; Hollopeter et al. [11]; Jin et al. [42]). C6 cells also disulphonic acid (DIDS), are inhibitors of nucleotide express the phospholipase (PL)C"-coupled P2Y ,P2Y , 1 2 hydrolyzing enzymes and are often used in studies of P2Y and P2Y receptors (Czajkowski et al. [41]; Nicholas 4 6 nucleotide-mediated signalling (Grobben et al. [21]). et al. [43]; Tu et al. [44]; Claes and Slegers [17]). Recently, Nevertheless, care must be taken for the interpretation we also demonstrated the presence of P2Y mRNA (Van of experimental data since it is also shown that cells can Kolen and Slegers [45]) implicating the expression of internalise some of these molecules (Claes et al., [22]). three ADP-activated receptors in these cells, i.e., P2Y 1, Therefore specific P2Y receptor antagonists, devel- P2Y and P2Y . Although 2MeSADP is reported as a 12 13 oped for therapeutical purposes, have to be used to potent P2Y ,P2Y and P2Y agonist, stimulation with 1 12 13 overcome the lack of specificity (Boeynaems et al. this compound inhibits AC, but induces no significant [23]; Lambrecht et al. [24]; Kam and Nethery [25]; Kim activation of PLC, indicating that the P2Y receptor is not et al. [26]; Xu et al. [27]). activated by ADP in cells grown in chemically defined medium (Grobben et al. [40]). This is confirmed by Czajkowski et al. [46], who showed that, in cells cultivated in the presence of fetal calf serum, ADP signalling is P2Y receptor expression on rat C6 glioma cells predominantly determined by the P2Y receptor. Howev- er, upon serum deprivation, expression of the P2Y Rat C6 glioma is a tumoral cell line of glial origin with receptor is decreased and the P2Y receptor becomes oligodendrocytic, astrocytic and neuronal progenitor the main activated receptor. Characterization of P2Y properties. Due to a point mutation in Fphosphatase receptor function is complicated by the fact that P2Y and tensin homologue deleted on chromosome ten_ and P2Y receptors have almost the same agonist profile (PTEN), the phosphatidylinositol 3-kinase (PI 3-K)/ (Table 1). The receptor antagonist N -(2-methyl- PKB signalling pathway is constitutively active and thioethyl)-2-(3,3,3-trifluoropropylthio)-",+-dichloromethy- contributes to the proliferative and invasive properties lene ATP (AR-C69931MX), often used as a specific of these cells (Kubiatowski et al. [28]; Roymans and P2Y antagonist, also blocks the P2Y receptor (Marteau 12 13 Slegers [29]; Grobben et al. [30]). In addition, cell et al. [47]). While the human and mouse P2Y receptor, proliferation is sustained by secreted growth factors like the P2Y , is more potently activated by 2MeSADP that stimulate growth factor receptors present on these than ADP, the rat P2Y receptor shows a higher cells. Such autocrine mechanisms are reported for selectivity for ADP (Fumagalli et al. [48]). In C6 cells, IGFR, bFGFR and PDGFR (Okumura et al. [31]; further distinction between the signalling of P2Y and Resnicoff et al. [32]; Strawn et al. [33]). P2Y receptors can be made by the use of PPADS, a 1 4 In C6 cells, an increase in cAMP by stimulation of P2Y antagonist without effect on P2Y ,and P ,P - 13 12 the "-adrenergic receptor ("-AR) or by addition of di(adenosine-5 ) tetraphosphate (Ap A), a P2Y antago- 4 13 membrane permeable cAMP analogues, e.g., dibutyryl nist that stimulates the P2Y receptor (Claes et al. [39]; 12 454 Purinergic Signalling (2006) 2:451–469 Grobben et al. [40]; Marteau et al. [47]). In addition, sitol 4,5-bisphosphate into the intracellular messenger recently synthesised PPADS derivatives pyridoxal-5 - inositol 1,4,5-triphosphate (IP ) and diacylglycerol phosphate-6-azo-(2-chloro-5-nitrophenyl)-2,4-disulphonate (DAG). Activation of PLC occurs by G !- and/or (MRS2211) and pyridoxal-5 -phosphate-6-azo-(4-chloro-3- G !-dependent mechanisms (Communi et al. [55]). nitrophenyl)-2,4-disulphonate (MRS2603) have no effect Besides signalling through G! subunits, intracellular 2+ on the P2Y receptor but antagonise the P2Y receptor Ca concentration is also affected by G"+ subunit- 12 13 2+ (Kim et al. [26]). dependent interaction with voltage-gated Ca chan- Although P2Y receptor expression in C6 cells de- nels. Several reports indicated modulation of K pends on the cultivation conditions (Czajkowski et al. currents and PLC" activation induced by distinct [46]), unpublished data of our laboratory revealed that domains of G"+ (Mirshahi et al. [56]). Co-expression induction of differentiation into astrocytes type II by studies performed in rat sympathetic neurons demon- dbcAMP (1 mM)- or (-)-isoproterenol (5 2M) does not strated that P2Y , P2Y and P2Y receptors trigger the 1 2 6 2+ + significantly alter the expression of P2Y receptors. closing of N-type Ca and M-type K channels, These observations are in accordance with previous whereas P2Y receptor stimulation also displayed studies on the expression of P2Y , P2Y , P2Y , P2Y , coupling to M-type K channels producing a less 1 2 4 6 2+ P2Y ,P2Y and P2Y receptors in glial cells and efficient inhibition of Ca currents. In rat brain cap- 12 13 14 primary astrocytes (Bianco et al. [49]; Fumagalli et al. illary endothelial cells, it was shown that the P2Y [48, 50]; Sasaki et al. [51]). The function of P2Y receptor receptor inhibits I and activates a G protein- Ca(N) expression in glial cells is still under investigation, but a coupled inward rectifier K (GIRK) channel. Interest- number of studies point to an important role in the ingly, stimulation of the P2Y receptor also induces a intercellular communication between astrocytes and K current that is rapidly followed by inactivation. neurons (Bezzi and Volterra, [52]). Another well Inhibition of I by P2Y receptor stimulation is Ca(N) 12 documented effect of extracellular ATP is induction also reported in PC12 cells while in HEK 293 this of reactive astrogliosis upon activation of ERK and inhibition is mediated by the P2Y receptor (Filippov cyclooxygenase (COX)-2 (Brambilla et al. [53]). et al. [57–60]; Simon et al. [61]; Wirkner et al. [62]). Similar to the observations made in C6 cells, From co-expression studies of P2Y receptors with functional responses of P2Y receptor subtypes in GIRK1 or GIRK2 in rat sympathetic neurons, it was microglial cells depend on cultivation conditions. In concluded that P2Y receptors activate GIRK channels N9 mouse brain microglia stimulation of expressed P2Y by the "+ subunits of G and inhibit these channels by i/o 2+ receptors induces Ca mobilization but only P2Y and the ! subunits of G (Filippov et al. [63]). 6 q P2Y receptor-mediated responses are increased upon P2Y-induced calcium release is followed by opening 2+ activation of microglia with lipopolysaccharide. The of voltage-independent Ca channels. Although this enhanced P2Y response is correlated with mRNA response is observed in a variety of cell types, the increase which was not the case for the P2Y physiological implications are miscellaneous. In this 2+ receptor-mediated Ca mobilization (Bianco et al. context, it has been reported that extracellular ATP 2+ [49]). Furthermore, stimulation of microglial P2Y induces a Ca wave that propagates through neigh- 12/13 receptors induces membrane ruffling and chemotaxis bouring astrocytes by GAP junctions (Suadicani et al. towards injured neurons through G protein-medi- [64]). In situations of increased neuronal activity or cell i/o 2+ ated activation of Rac (Honda et al. [54]). The damage, ATP stimulates a Ca -dependent proton- observations made in astrocytes and microglial cells efflux from astrocytes. Acidification of the extracellular emphasise the importance of P2Y receptors in brain environment serves as a negative feedback mechanism signalling and identify these receptors as putative for neurotransmitter release, but also increases blood targets in defective neurotransmission, neuroimmune flow by vasodilatation in cerebral arterioles (Dienel and 2+ functioning, cell survival and cell proliferation in Hertz [65]; Dixon et al. [66]). Although Ca signalling response to oxidative stress and brain injury. is observed in a variety of cell types, the time dependence of the response is cell type specific. This is especially the case for the P2Y receptor which 2+ P2Y receptor-activated signalling cascades triggers persistent or transient Ca responses when it is expressed in human 1321N1 astrocytoma or C6 glioma Second messengers cells, respectively (Czajkowski et al. [41]; Sellers et al. [67]). A recent study also revealed that, in glial cells, 2+ P2Y receptors are generally linked to PLC activation prolongation of the P2Y receptor-induced Ca re- + + that catalyses the rapid hydrolysis of phosphatidylino- sponse is regulated by interaction with the Na /H Purinergic Signalling (2006) 2:451–469 455 exchanger regulatory factor type-2 which determines ceptors is sometimes sufficient to exert a significant the signalling pathways that are ultimately activated in inhibitory action towards PKA. Such a response is different cell types (Fam et al. [68]). Indeed, while reported in microglial cells where ATP and ADP transient P2Y receptor signalling increases prolifera- binding to P2Y receptors mediate chemotaxis by 1 12/13 tion in C6 cells, sustained signalling triggers apoptotic PKA-dependent translocation of "1 integrins to ruf- cascades in 1321N1 astrocytoma cells (Czajkowski et al. fling regions of the cell (Nasu-Tada et al. [76]). [46]; Sellers et al. [67]). Despite the fact that cells express a myriad of A well-known response to PLC-generated DAG and different GPCRs and downstream acting regulators, 2+ IP /Ca is the activation of classical PKCs that are receptor stimulation promotes rapid and specific involved in rapid internalisation and desensitisation of responses. In addition, multiple GPCRs, sharing the GPCRs through phosphorylation of residues localised same second messenger cascade, can induce different in their cytoplasmic tail. In this regard, PKC"Iis cellular events in one cell type indicating that GPCR reported to attenuate phosphatidylinositol (PI)-hydro- signal propagation requires physical interactions in a lysis induced by P2Y and P2Y receptors in endothelial defined cellular compartment. An example of spatial 1 2 cells (Chen and Lin [69]). In astrocytes, high frequency organised signalling is the "-arrestin-dependent target- stimulation of the P2Y receptor by repeated addition ing of an activated receptor into clathrin-coated vesicles of ATP causes rapid suppression of the P2Y receptor- or enrichment in membrane microdomains (lipid rafts) 2+ induced Ca response. This phenomenon, observed as formed by cholesterol and sphingolipids (Anderson 2+ Ca oscillations, is mediated by protein kinase C- [77]; DeFea et al. [78]). Modulation of receptor dependent phosphorylation of Thr339 in the carboxy- function by rafts is confirmed for an increasing number terminus of the P2Y receptor (Fam et al. [70]). Besides of GPCRs including P2Y receptors (Anderson [77]; modulation of receptor responsiveness, PKC signalling Ostrom and Insel [79]). In endothelial cells, it is also affects long term effects. In the human osteoblastic reported that P2Y receptor-induced vasodilatation is HOBIT cell line, ATP increases expression of the early abolished by disruption of caveolae with methyl-"- growth response protein-1 by a mechanism that requires cyclodextrin (Kaiser et al. [80]). In C6 glioma cells, 2+ aCa -independent PKC isoform (Pines et al. [71]). In signalling by P2Y and 5-hydroxytryptamine (HT) 2 2A vascular smooth muscle cells, UDP stimulates cell cycle receptors is attenuated after knock-down of caveolin-1 progression by a PLC- and PKC%-dependent cascade by si-RNA. Moreover, interaction between the 5-HT 2A (Hou et al. [72]). The same isoform is involved in ATP- receptor and caveolin-1 facilitates its interaction with mediated mitogenic signalling in astrocytes, but in these G! . Since P2Y receptor mRNA is downregulated by q 2 cells PKC% activation does not involve PLC but caveolin-1 knock-down, further studies are required to requires PLD-dependent choline formation (Neary demonstrate localisation of P2Y receptors in caveolae et al. [73]). of C6 cells (Bhatnagar et al. [81]). In addition to PLC-coupled receptors, a growing number of P2Y receptors have been shown to affect the activity of AC. Besides the existence of indirect Small GTPases as molecular switches mechanisms linked to an increase in cAMP (discussed in Communi et al. [55]), only the P2Y receptor is The processing of extracellular stimuli by GPCRs often involves signalling by second messengers directly coupled to activation of AC and PLC while 2+ P2Y , P2Y and P2Y receptors negatively affect (cAMP, DAG, Ca ) towards small GTPases and/or 12 13 14 cross-talk with tyrosine kinases. G protein-coupled cAMP synthesis (Chambers et al. [13]; Communi et al. [10]; Hollopeter et al. [11]; Zhang et al. [12]). receptor signalling via PLC" induces formation of 2+ DAG and IP ,Ca mobilisation and activation of Adenylate cyclase-dependent signalling is often me- diated by the cAMP-regulated kinase PKA. Stimula- PKC ultimately leading to activation of proline-rich tyrosine kinase 2 (Pyk2). Pyk2 cooperates with Src to tion of the P2Y receptor with ATP is shown to activate human monocyte-derived dendritic cells by recruite Grb2 and SOS, a guanine nucleotide exchange increased cAMP/PKA signalling (Wilkin et al. [74]). In factor (GEF) that activates Ras (Lev et al. [82]). Such a mechanism is reported for Ras-dependent ERK acti- bovine adrenocortical fasciculate cells, ADP and ATP increase cortisol production through PKA activation vation induced by the protease-activated receptor-1 in astrocytes (Wang and Reiser [83]). In PC12 cells, by an as yet unidentified G protein-coupled P2Y receptor (Nishi et al. [75]). Although in unstimulated stimulation of the P2Y receptor also triggers tyrosine phosphorylation of Pyk2, but further signalling to Ras cells the cytosolic cAMP concentration is already low, its further decrease by G protein-coupled re- involves EGFR transactivation by Src (Soltoff et al. i 456 Purinergic Signalling (2006) 2:451–469 [84]). Tyrosine kinase-dependent Ras signalling is also Honda et al. [54]). Stress fibre formation in vascular reported for G protein-coupled receptors, but this smooth muscle cells is reported to be mediated by proceeds through G"+ subunit-mediated activation of RhoA/ROCK signalling that becomes activated upon PI 3-K+ and Shc (Ellis et al. [85]; Lopez-Ilasaca et al. stimulation of P2Y ,P2Y , P2Y and P2Y receptors 1 2 4 6 [86]). (Sauzeau et al. [99]). In the latter study, information GEFs can also be regulated in a tyrosine kinase- concerning the signalling towards RhoA is lacking. In independent manner that proceeds through direct a more recent study on endothelial cells, transactiva- 2+ activation by cAMP, DAG and Ca or by interaction tion of VEGFR upon P2Y receptor stimulation and with G! subunits as observed for the G protein- recruitment of the RhoGEF Vav is shown to be a q/11 mediated activation of RhoA (Bhattacharya et al. [87]; possible mechanism to initiate RhoA-mediated cell Bos [88]; Lutz et al. [89]; Walker et al. [90]). adhesion (Seye et al. [100]). P2Y receptor signalling towards GTPases is involved Although several P2Y receptors activate RhoA, in short term responses, such as stress fibre formation or downstream signalling and physiological consequences modulation of cell adhesion, but also in long term are determined by celltype specific mechanisms lead- responses like cell proliferation. Mitogenic Ras-depen- ing to diverse responses. dent P2Y responses are reported for C6 and HEK293 cells where Ras is implicated in P2Y receptor-depen- ERK signalling dent signalling to the ERK pathway (Gao et al. [91]; Tu et al. [44]). On the other hand, increased prolifer- Several GPCRs are coupled to enhanced proliferation ation of C6 cells by the P2Y receptor proceeds by multiple signal transduction pathways that phos- independently of Ras, but requires RhoA-dependent phorylate ERK. Activation of this kinase requires Ras activation of ERK and Rho-associated coiled-coil- or GTPases of the RhoA family and is often modulated containing protein kinase (ROCK) (Grobben et al. by second messenger-activated pathways, although [40]; Van Kolen and Slegers, unpublished data). cross-talk with growth factor receptors also triggers Interestingly, when the P2Y receptor is expressed ERK signalling. in CHO cells it activates ERK and RhoA/ROCK In neurons G protein-mediated activation of AC by independent mechanisms (Soulet et al. [92]). increases ERK phosphorylation by a PKA/Rap1/B- Another example of cross-talk between P2Y recep- Raf cascade. In contrast, induction of cAMP synthesis tors and GTPases is observed in blood platelets. As decreases ERK phosphorylation in C6 cells and mentioned above, release of ADP and subsequent P2Y astrocytes by a negative action of PKA on the Ras/c- and P2Y receptor binding is essential for collagen- Raf1 interaction, or by Rap1-mediated inhibition of c- induced platelet aggregation. A crucial step for imme- Raf1. These observations led to the hypothesis that an diate and sustained aggregation of platelets is the increase in cAMP stimulates MEK/ERK signalling in activation of Rap1 that increases the affinity between B-Raf expressing cells but inhibits this cascade in B- integrin ! " and fibrinogen. Knock-out studies Raf negative cells (Dugan et al. [101]). In both cases, IIb 3 revealed that ADP-induced GTP loading of Rap1 PKA activation has a central role and mediates its proceeds through both G and G signalling by P2Y effects through Src and Rap1 activation (Stork and i q 12 and P2Y receptors, respectively. The mechanism Schmitt [102]). Although the majority of cAMP- initiated by the P2Y receptor is shown to be PI 3- dependent effects can be explained by this hypothesis, K-dependent while P2Y -mediated activation of Rap1 a few exceptions are reported. In some B-Raf positive 2+ requires Ca mobilisation (Woulfe et al. [93]; Greco cells, an increase in cAMP is shown to inhibit B-Raf, et al. [94]; Larson et al. [95]; Lova et al. [96, 97]). suggesting that regulation of this kinase by cAMP also Stimulation of the P2Y receptor also contributes to depends on other cell type specific factors. One model 2+ platelet shape changes by a Ca -independent path- suggests the involvement of 14-3-3 proteins acting as way. RhoA and its effector ROCK are activated by scaffolding proteins to shield B-Raf and Raf1 from ADP through G protein-dependent signalling of PKA phosphorylation (Qiu et al. [103]). Other studies 12/13 the P2Y receptor and contribute to rapid actin indicated that regulation of ERK by cAMP involves polymerization and shape changes (Paul et al. [98]). multiple cell type specific mechanisms. In COS cells Signalling towards Rho GTPases is also important in overexpressing "-AR1 or "-AR2, stimulation of these other systems. In brain, ATP and ADP induce receptors activate AC through a G protein-dependent membrane ruffling and chemotaxis of microglial cells mechanism as expected. However, PKA also phos- through G protein-dependent activation of Rac upon phorylates these receptors and induces a switch from stimulation of P2Y receptors (Sasaki et al. [51]; G to G protein binding to "-AR resulting in 12/13 s i/o Purinergic Signalling (2006) 2:451–469 457 activation of ERK upon receptor stimulation (Martin thrombin-induced MEK and ERK activation indepen- et al. [104]). Modulation of the ERK cascade by cAMP dently of Ras or Raf (Nadal-Wollbold et al. [119]). can also occur independently of PKA. In this context, G protein-coupled receptors that are not linked to i/o cAMP binds Epac1 or Epac2, Bexchange protein PLC activation can also modulate mitogenic signalling directly activated by cAMP,’’ GEFs that activate through G"+-dependent activation of PI 3-K+. Signal- Rap1 and Rap2 (de Rooij et al. [105, 106]; Kawasaki ling from PI 3-K+ to ERK proceeds through Shc/Grb2/ et al. [107]). Several examples of G protein-mediated SOS/Ras (Lopez-Ilasaca et al. [86]). An increasing activation of ERK through Epac are reported number of reports point to the involvement of PKCK in (Laroche-Joubert et al. [108]; Lin et al. [109]). Another G protein-dependent phosphorylation of ERK. The PKA-independent mechanism of ERK phosphoryla- first observation was made in CHO cells where tion is the G "+/Src-mediated activation of Ras stimulation of the LPA receptor triggers MEK/ERK (Schmitt and Stork [110]). signalling via a PI 3-K+-dependent activation of PKCK G and some G protein-coupled receptors activate not abrogated by transfection with dominant negative q i/o PLC" and trigger formation of IP and DAG, resulting Ras (Takeda et al. [120]). In addition, a recent report 2+ 2+ in Ca release and PKC activation, respectively. Ca indicated that angiotensin II-induced ERK activation increase can activate ERK through Pyk2 that activates in rat vascular smooth muscle cells requires interaction 2+ Ras as mentioned above. Otherwise, Ca -dependent between Ras and PKCK (Zhao et al. [121]). PKCK- modulation of Ras activity is also mediated by Ras dependent activation of ERK is mediated by inter- guanine nucleotide-releasing factor (RasGRF), a GEF action with MEK, a property shared by other PKC 2+ that contains Ca - and DAG-binding domains, isoforms (Scho ¨ nwasser et al. [122]), or by regulation of 2+ (Ebinu et al. [111]) or by Ca /calmodulin-dependent Raf1. Studies performed in rat embryonic hippocampal kinases CaMK-II and CaMK-IV (reviewed in Agell cells indicated that PKCK can phosphorylate the Raf et al. [112]and Walker et al.[90]). Increase of kinase inhibitory protein (RKIP) resulting in dissocia- intracellular calcium and DAG formation also results tion of the Raf1/RKIP complex (Corbit et al. [123]). In in activation of cPKCs while DAG formation alone is addition, co-immunoprecipitation experiments in COS sufficient to activate nPKCs. Increase in PKC activity cells showed that modulation of c-Raf1 by PKCK is modulates the ERK cascade through Ras by inhibition also regulated by 14-3-3 scaffolding proteins (Van Der of RasGAPs and/or stimulation of RasGEFs. In Hoeven et al. [124]). addition, PKC can activate Raf independently of Ras. Initial studies concerning P2Y receptor-mediated Indeed, it is shown that PKC! phosphorylates Raf at activation of ERK were made in astrocytes where this Ser499 (Kolch et al. [113]). However, mutation of this cascade was shown to be involved in cell proliferation serine residue into alanine does not affect Raf activity and process elongation (Neary and Zhu [125]; King in response to phorbol esters (Yip-Schneider et al. et al. [126]). [114]). More convincing data were obtained when Although ATP triggers pertussis toxin insensitive 2+ constitutively active PKC was expressed in rat 6 IP and Ca responses in astrocytes, these are not fibroblasts. These cells display Ras-independent sig- required for the signalling towards ERK which nalling towards ERK by direct phosphorylation of Raf depends on rapid membrane translocation of PKC% by PKC(. Since activation of Ras is required in several upon phosphatidylcholine hydrolysis by PLD (Neary systems this interaction is cell type-dependent (Cacace et al. [73]). In PC12 cells, stimulation of the P2Y re- et al. [115]; Ueffing et al. [116]). Direct phosphoryla- ceptor also induces PKC%-dependent ERK phosphor- 2+ tion of Raf by PKC is also involved in ERK activation ylation, although this mechanism requires Ca and by the G protein-coupled leukotriene (LT)D receptor Pyk2 for the association of Shc and Grb2 to the i 4 in intestinal epithelial cells. Although stimulation of receptor and for subsequent activation of SOS/Ras/ this receptor also triggers a parallel PKC-independent Raf/MEK/ERK (Soltoff et al. [84]). Many reports activation of Ras, transfection experiments confirmed showed that P2Y receptor-mediated ERK signalling that Ras is dispensable for LTD receptor-mediated requires PKC activation (Graham et al. [127]; Huwiler ERK activation (Paruchuri et al. [117]). When consti- and Pfeilschifter [128]; Erlinge [129]), but a PKC- tutive active point mutants of PKC!, PKC% and PKC( independent mechanism is reported in thyroid FRTL- were introduced in COS cells, only PKC% activated the 5 cells (Tornquist et al. [130]). In 1321N1 astrocytoma ERK cascade (Ueda et al. [118]), indicating that cells, stimulation of the P2Y receptor with UDP acti- involvement of PKC isoforms in ERK signalling vary vates PKC!, ( and K which are correlated with ERK among different cell types. This is also confirmed by the phosphorylation (Kim et al. [131]). Although the use of observation that, in platelets, cPKCs are involved in general PKC inhibitors 3-[1-(dimethylaminopropyl) 458 Purinergic Signalling (2006) 2:451–469 indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride dependent activation of p38 and ERK1/2 (Berenbaum (GF109203X) and 12-(2-cyanoethyl)-6,7,12,13-tetrahy- et al. [136]). In primary astrocytes, P2Y receptor- dro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)- mediated ERK activation by ATP is shown to induce carbazole (Go ¨ 6976) diminished ERK signalling, the reactive astrogliosis, a phenomenon that occurs upon lack of specificity of these compounds makes it dif- brain injury and is characterised by astroglial prolifer- ficult to determine the contribution of each of these ation, cellular hypertrophy and up regulation of GFAP. PKC isoforms in the mechanism of ERK activation This effect is mediated by an ERK-dependent increase (Way et al. [132]). in the expression of COX-2 (Brambilla et al. [53]). In Enhanced proliferation by a P2Y receptor-mediated C6 cells, P2Y and P2Y receptor-induced activation 12 2 stimulation of the ERK pathway has been reported in of ERK is coupled to an enhanced cell proliferation, a large number of cell types such as human mesangial while a negative modulation of GFAP synthesis by the cells, vascular smooth muscle cells and primary astro- P2Y receptor is reported (Claes et al. [39]; Tu et al. cytes (Huwiler and Pfeilschifter [128]; Harper et al. [44]; Van Kolen and Slegers [45]). These differences [133]). Transient ERK activation by P2Y , P2Y or are probably due to the fact that in C6 cells induction 1 2 P2Y receptor stimulation also increases cell prolifer- of GFAP expression is not correlated with an en- ation in C6 cells indicating that activation of several hanced proliferation but requires growth arrest. P2Y receptor subtypes can converge into the same In summary, most P2Y receptors are coupled to physiological response (Table 2) (Tu et al. [44]; Claes ERK phosphorylation, but the signalling mechanism et al. [39]; Czajkowski et al. [46]). and the physiological effect of this pathway are cell type In addition to mitogenesis, P2Y receptor signalling specific and are determined by the cellular context. towards ERK elicits other physiological processes including cell survival, inflammation and reactive PI 3-K/PKB signalling gliosis. In human lung microvascular endothelial cells, hyperoxia-induced release of ATP results in cell PKB/Akt is involved in a large variety of cellular survival through ERK and PI 3-K signalling cascades processes including glucose metabolism, mitogenesis, activated by P2Y and/or P2Y receptors, while 2 6 stimulation of the ERK cascade by the P2Y receptor differentiation, survival and motility (Brazil et al. [137]). This member of the AGC protein kinase protects 1321N1 astrocytoma cells from TNF!-induced apoptosis (Ahmad et al. [134]; Kim et al. [131]). Rapid superfamily is recruited to the plasmamembrane upon PI 3-K-mediated PIP3 formation, but is also controlled ERK1/2 and p38 MAPK activation plays an important role in P2Y receptor-dependent primary granule in a PI 3-K-independent, but calmodulin-dependent, 2+ fashion upon intracellular Ca mobilisation by stimu- release from human neutrophils (Meshki et al. [135]). A similar phenomenon is observed in articular chon- lation of neuronal NMDA receptors (Cantley [138]; Leevers et al. [139]; Woodgett [140]; Yano et al. [141]). drocytes where ATP acts as a pro-inflammatory medi- ator by increasing arachidonic acid production and Modulation of PKB activity is reported for a variety release of prostaglandin E through a P2Y receptor- of GPCR ligands including adrenergics, cannabinoids, 2 2 Table 2 G protein-dependent modulation of ERK and PKB signalling cascades in C6 cells. G protein ERK PI 3-K/PKB Effect 2+ P2Y G j PLC-PKC-Ca -Ras. , Attenuation of PI 3-K Proliferation 1 q activated by growth factors 2+ P2Y G j PLC-PKC-Ca -Ras. – Proliferation 2 q P2Y G j RhoA-PKC-Raf-MEK j PI 3-K/PKB Proliferation/inhibition of 12 i astrocytic differentiation 2OR G j FGF transactivation – Proliferation "-AR G , Transient inhibition , Transient inhibition Growth arrest/astrocytic dependent on cAMP by cAMP differentiation CB G , Sustained inhibition , Sustained inhibition Growth arrest/apoptosis Transient ERK activation by P2Y (Czajkowski et al. [46]), P2Y inhibits ERK and PKB concomitant with induction of differenti- 1 2 (Tu et al. [44]), P2Y (Grobben et al. [40]) and 2 opioid receptors ation (Wang et al. [149]; Van Kolen and Slegers [45]). Inhibition of (2OR) (Belcheva et al. [198]) enhances cell proliferation while these pathways by cannabinoids (CB) is sustained and induces stimulation of the "-adrenergic receptor ("-AR) transiently apoptosis (Ellert- Miklaszewska et al. [184]). Purinergic Signalling (2006) 2:451–469 459 carbachol, glutamate, histamine, nucleotides and ATP or UTP activates PKB by a PDK-1-dependent thrombin (Dickenson [142]; Franke et al. [143], mechanism while, in C6 cells, ADP activates PI 3-K/ Iacovelli et al. [144] Murga et al. [145]; Sanchez et al. PKB by the G protein-coupled P2Y receptor but i 12 [146]). Due to the existence of multiple phosphoinosi- inhibits PI 3-K by stimulation of the G /G protein- q 11/12 tide-dependent cascades, regulation of PKB signalling coupled P2Y receptor (Table 2) (Van Kolen and by GPCRs varies among the studied systems. Slegers [45]; Czajkowski et al. [46]; Huwiler et al. In HEK293 cells, stimulation of "-AR with [159]). Although most effects of P2Y-mediated activa- (-)-isoproterenol activates PKB via G "+, Src, Ras and tion of PI 3-K signalling are known to be related to cell PI 3-K (Schmitt and Stork [110]; Bommakanti et al. proliferation, differentiation and survival, this signal- [147]) while activation of AC by G! exerts differential ling cascade is also involved in other processes. In this effects on PKB activity. In cells expressing Epac, cAMP regard, it can be mentioned that P2Y receptor- activates PI 3-K/PKB via Rap1 while, in other cells, mediated PI 3-K/PKB activation modulates prolifera- cAMP activates PKA that exerts a negative action on PI tion and differentiation of C6 cells, but also plays an 3-K and PKB (Mei et al. [148]; Wang et al. [149]). important role in ADP-induced platelet aggregation G protein-mediated activation of PKB can occur (Van Kolen and Slegers [45]; Czajkowski et al. [46]; through the coupling of the G"+ subunit to the catalytic Chen et al. [160]; Kim et al. [161]). subunit of PI 3-K or via growth factor receptor trans- activation. Although only p110+ was initially reported to be activated by G"+ subunits, this feature is also P2Y receptor-integrated G protein-coupled receptor observed for the p110" isoform (Kurosu et al. [150]; and receptor tyrosine kinase signalling cascades Stoyanov et al. [151]). This mechanism is reported in Vero cells where stimulation with LPA activates Ras G protein-coupled receptor cross-talk upon increase in p110" lipid kinase activity (Yart et al. [152]). G protein-mediated transactivation of Complementary to vertical downstream signalling upon growth factor receptors is reported in HaCaT, A-431, GPCR stimulation, these receptors also mediate lateral and HEK293 cells where stimulation of the angioten- signalling by cross-talk with other receptors (reviewed in sin type I receptor by mechanical stress induces Cordeaux and Hill [162]). In human platelets, it was transactivation of EGFR leading to activation of the reported that P2Y receptor activation potentiates 2+ PI 3-K/PKB cascade and protection of these cells from P2Y receptor-mediated Ca signalling, while the apoptosis (Kippenberger et al. [153]). P2Y receptor negatively regulates this action (Hardy In 1321N1 astrocytoma cells, PLC" activation by the et al. [163]). In renal mesangial cells, P2Y receptors G protein-coupled muscarinic M receptor also trig- activated by ATP and UTP induce a rapid desensitisa- q 3 gers PI 3-K activation through ErbB3 transactivation, tion of the sphingosine-1-phosphate (S1P) receptor by 2+ but this mechanism requires Ca mobilisation (Tang PKC-dependent phosphorylation (Xin et al. [164]). A et al. [154]). In contrast, some reports showed an more complex interplay is observed between P2Y inhibitory pathway from G protein-coupled receptors receptors and 5-HT receptor subtypes. Studies per- towards PI 3-K by direct interaction between G!- formed in CHO cells stably expressing 5-HT recep- 1A subunits released from heterotrimeric G proteins and tors revealed that the responsiveness of this receptor is p110!, as reported for the ! -AR in rat-1 fibroblasts reduced by a PLD/PKC-dependent phosphorylation 1A (Ballou et al. [155, 156]), or by inhibition of insulin upon short (<5 min) pre-treatment with ATP, while receptor substrate-1-associated PI 3-K activity in the agonist efficacy of the overexpressed 5-HT 1B 1321N1 astrocytoma cells by carbachol, histamine or receptor is not altered. Alternatively, longer treatment thrombin. These observations reveal opposing effects with ATP alone attenuates 5-HT signalling by a 1B of muscarinic receptor stimulation on PI 3-K activity mechanism that requires activation of phospholipase mediated by insulin and ErbB3 receptors in these cells A (PLA ) (Berg et al. [165]). Furthermore, stimulation 2 2 (Batty et al. [157]). of P2Y receptors can also modulate the release of Modulation of PI 3-K/PKB signalling is also reported transmitter molecules, including dopamine, glutamate for a few P2Y receptors. In bovine adventitial fibro- and serotonin (Bezzi and Voltera [52]; Krugel et al. blasts, ATP is shown to induce proliferation through [166]; Nedergaard et al. [167]). A recently discovered parallel but independent ERK and PI 3-K signalling mechanism of GPCR cross-talk is the assembly of a cascades that contribute to mTOR and p70S6K phos- heteromeric receptor complex displaying the pharmaco- phorylation (Gerasimovskaya et al. [158]). In rat logical profile of one receptor and the signalling proper- mesangial cells, stimulation of the P2Y receptor with ties of the other. Such an interaction is reported in 2 460 Purinergic Signalling (2006) 2:451–469 HEK293 cells overexpressing A and P2Y receptors. Pyk2, probably by its proline-rich putative SH3 1 1 The heteromeric A -P2Y receptor complex inhibits binding sites (PXXP). This interaction is implicated 1 1 AC through G protein, but displays P2Y receptor- in P2Y receptor-induced transactivation of EGF, i/o 1 2 like pharmacological properties (Yoshioka et al. [168]). PDGF and VEGF receptors (Liu et al. [176]; Seye et al. [100]). Src inhibition abolishes growth factor P2Y receptor-mediated transactivation receptor transactivation and ERK phosphorylation. Although the rat P2Y receptor lacks PXXP motives, Many studies reveal that GPCRs and growth factor tyrosine kinase-dependent activation of ERK upon receptors share a number of signalling modules P2Y receptor stimulation is reported in a few rat cell (e.g., Raf/MEK/ERK, PI 3-K/PDK/PKB) to transduce lines, including C6 and PC12 cells (Soltoff et al. [84]; their effects. In the past decade, it has become clear Tu et al. [44]). In the latter cases, P2Y receptor- that the signalling pathways of both receptor systems dependent activation of Pyk2 is mediated by PKC and 2+ are interconnected. Stimulation of a GPCR can induce Ca suggesting that the PXXP sequence is dispens- a rapid tyrosine phosphorylation of RTKs. This trans- able for P2Y receptor-induced tyrosine phosphoryla- activation mechanism is reported for many GPCRs tion of Pyk2 and downstream signalling towards ERK. and proceeds through the G"+ subunit-dependent Moreover, P2Y mutants lacking PXXP-motives are activation of Src. Src in turn activates RTKs by still able to activate ERK demonstrating the existence phosphorylation of specific tyrosines located in their of other pathways towards phosphorylation of ERK intracellular domains or induction of matrix metal- (Liu et al. [176]). Observations made in human loproteases-dependent release of growth factor recep- endothelial cells, where UTP-induced signalling to 2+ tor ligands, e.g., release of heparin-bound EGF ERK was shown to depend on Ca ,PKC and (Luttrell and Luttrell [169]). integrin-mediated cell anchorage, already pointed to Another target for signal integration of GPCRs and a pathway distinct from the classical Ras/Raf/MEK/ RTKs are docking proteins. Although these proteins ERK cascade (Short et al. [177]). Human and mouse contain phospho-tyrosine binding domains that inter- P2Y receptors contain a RGD sequence which allows act with phosphorylated tyrosine residues of RTKs, activation of ERK by interaction with ! " /" integrins V 3 5 stimulation of GPCRs can induce growth factor followed by G protein coupling. Since these proteins receptor-independent phosphorylation of docking pro- also mediate cell adhesion and chemotaxis, the ob- teins by Src (Bisotto and Fixman [170]). served P2Y /! " /" -interaction also points to a possi- 2 V 3 5 In addition to GPCR-dependent phosphorylation of ble function of P2Y receptors in inflammatory RTKs, the opposite activation mechanism is also responses (Erb et al. [178]). reported. Binding of PDGF to its cognate receptor It is clear that, in analogy with other GPCRs, cross- induces association of PDGFR with the G protein- talk between P2Y and growth factor receptors may coupled S1P receptor. Subsequently, Src is recruited to occur at different levels of the signal transduction this complex by G"+ subunits and phosphorylates pathway depending on receptor subtypes and on the Grb-2 associated binder-1 resulting in dynamin studied system. For the P2Y receptor, additional II-induced Bpinching off’’ of vesicles involved in transactivation mechanisms are facilitated by the endocytosis of PDGF-S1P signalling complexes and presence of signalling motives (e.g., PXXP or RGD) subsequent activation of ERK1/2 (Waters et al. [171]). that allow direct interaction with other signalling Cross-talk between RTKs and P2Y receptors is components (Src, integrins). reported in Mu ¨ ller glial cells where ATP exerts its mitogenic effect through transactivation of EGF and PDGF receptors resulting in ERK-dependent en- P2Y receptor-activated signal transduction pathways hanced proliferation. In these cells, ATP-induced in C6 glioma cells activation of ERK was abolished by treatment with the RTK autophosphorylation inhibitor tyrphostin As mentioned above, the final outcome of nucleotide- (AG1478) (Milenkovic et al. [172]). In rat striatal mediated signalling is influenced by ecto-enzymes astrocytes, ATP and bFGF activate ERK and induce (Claes and Slegers [17]; Czajkowski and Baranska astrogliosis by a mechanism that is insensitive to [18]; Grobben et al. [21, 179]). ATP and ADP hy- RTK inhibition (Abbracchio et al. [173]; Bolego et al. drolysis to adenosine results in growth inhibition by a [174]; Neary et al. [175]). More recently, mechanistic mechanism that is not yet fully understood. When studies performed in 1321N1 astrocytoma cells reveal nucleotide hydrolysis is prevented, ATP, ADP and that the human P2Y receptor interacts with Src and Ap A (in particular Ap A and Ap A) increase cell 2 n 3 4 Purinergic Signalling (2006) 2:451–469 461 2+ proliferation more than two-fold. Stimulation with activates ERK through a Ca -dependent mechanism 2MeSADP, a P2Y agonist not hydrolysed by the (Czajkowski et al. [46]), likely by a similar mechanism ecto-enzymes present on the plasma membrane of C6 as reported for the P2Y receptor (Tu et al. [44]). In cells, also results in growth enhancement and inhibi- addition, it has been shown that ADP can activate tion of "-AR-induced differentiation into astrocyte ERK by stimulation of the P2Y receptor through a type II (Claes et al. [39]; Van Kolen and Slegers [45]). RhoA- and PKC-dependent pathway that does not 2+ The pathways involved in the P2Y receptor-dependent require Ca , Ras or tyrosine kinase activation 2+ effects on growth and differentiation of these cells are (Grobben et al. [40]). The fact that Ca removal does presented in Figure 1. not affect P2Y receptor-mediated ERK activation Nucleotides stimulate several purinergic receptors excludes the involvement of cPKCs. Stimulation of the that activate the ERK cascade by at least two distinct P2Y receptor does not induce PI-turnover, but mechanisms. The P2Y receptor, stimulated by UTP nPKCs might be involved since alternative activation and ATP, enhances ERK phosphorylation through a mechanisms, based on Ser/Thr and Tyr phosphoryla- PLC"/PKC/Ras/Raf/MEK cascade that is attenuated tion, have been reported (Steinberg [180]; Parekh et al. 2+ by inhibition of tyrosine kinases and Ca chelation by [181]). Data from our laboratory suggest an important 2+ BAPTA-AM (Tu et al. [44]). The Ca -dependence of role for PKCK in P2Y receptor-dependent activation the P2Y receptor-mediated activation of ERK sug- of ERK. The fact that no cross-talk between ERK and gests the involvement of a cPKC (!, "I, "II or +). It is PI 3-K is observed in C6 cells indicates that PKCK also shown that ADP stimulates the P2Y receptor and exerts its actions independently of PI 3-K via a RhoA- Figure 1 Overview of P2Y receptor-mediated signalling cascades in C6 cells. Green and red lines represent stimulatory (green arrows) Figure 1 Overview of P2Y receptor-mediated signalling cascades lation also inhibits cAMP-dependent induction of differentia- and inhibitory (red squares) actions respectively. Dashed lines are incomplete characterised pathways. P2Y receptor stimulation in C6 cells. Green and red lines represent stimulatory (green tion by reactivation of PKB which requires Src/Pyk2 complex enhances ERK-dependent proliferation through a PLC-dependent pathway while P2Y receptor stimulation enhances cell arrows) and inhibitory (red squares) actions respectively. Dashed formation and Rap1 activation. Formation of the Src/Pyk2 proliferation by RhoA- and PKCK-dependent activation of ERK (Claes et al. [39]; Grobben et al. [40]; Tu et al. [44]; Van Kolen 2+ lines are incomplete characterised pathways. P2Y receptor complex requires Ca and PLD2 which is constitutively active and Slegers, unpublished data). P2Y receptor stimulation also inhibits cAMP-dependent induction of differentiation by reactivation stimulation enhances ERK-dependent proliferation through a (Claes et al. [22]; Van Kolen and Slegers [45]; Van Kolen et al. 2+ of PKB which requires Src/Pyk2 complex formation and Rap1 activation. Formation of the Src/Pyk2 complex requires Ca and PLD2 PLC-dependent pathway while P2Y receptor stimulation [185]). Cyclic AMP-dependent inhibition of PKB and ERK is which is constitutively active (Claes et al. [22]; Van Kolen and Slegers [45]; Van Kolen et al. [185]). Cyclic AMP-dependent inhibition enhances cell proliferation by RhoA- and PKCK-dependent suggested to depend on inhibition of Rap1 (Wang et al. [149]). of PKB and ERK is suggested to depend on inhibition of Rap1 (Wang et al. [149]). The negative modulation of PI 3-K by the P2Y activation of ERK (Claes et al. [39]; Grobben et al. [40]; Tu The negative modulation of PI 3-K by the P2Y receptor is only et al. [44]; Van Kolen and Slegers, [199]). P2Y receptor stimu- displayed in the presence of serum (Czajkowski et al. [46]) 12 462 Purinergic Signalling (2006) 2:451–469 dependent mechanism (Grobben et al. [40]; Van Kolen inhibition of PKB while recovery of this activity is and Slegers, [199]). Although P2Y receptors use required to prohibit cell death. This hypothesis is different mechanisms to activate ERK, they all con- confirmed by a recent study where sustained inhibition verge to increased cell proliferation by enhanced of PI 3-K/PKB by cannabinoids is shown to induce synthesis of c-Myc, c-Jun and c-Fos (Zhang et al. apoptosis in C6 cells (Table 2) (Ellert-Miklaszewska [182]). Progression through the G1/S phase of the cell et al. [184]). Taken together, P2Y receptor stimula- Kip cycle is due to a decreased expression of p27 and tion inhibits cAMP-dependent induction of differenti- increased expression of cyclinD. ation by a transient increase in PI 3-K/PKB activity. 2+ While stimulation of ERK signalling by P2Y recep- Ca chelation inhibits the basal PKB activity and tors has been known for several years, the coupling with P2Y receptor-mediated increase in PKB phosphoryla- PI 3-K activation was discovered more recently. When tion. Although C6 cells also express the P2Y receptor, C6 cells are grown in the presence of serum, P2Y stimulation with UTP does not enhance the activity of receptor signalling predominates and is shown to PI 3-K/PKB, which may be explained by a differential inhibit PI 3-K (Czajkowski et al. [46]). Upon serum coupling to G protein subtypes. P2Y receptor-mediat- deprivation, P2Y receptor expression decreases while ed signalling proceeds through G proteins while the 1 q P2Y becomes the main ADP-stimulated receptor activation of PDK is G protein-dependent (Table 2) 12 i that enhances the activity of PI 3-K by a G protein- (Tu et al. [44]; Huwiler et al. [159]). The lack of G i i dependent mechanism. These observations demon- protein coupling of the P2Y receptor in C6 cells might strate that, in addition to autocrine growth factor be a consequence of compartimentalisation into cav- receptor signalling, the constitutive PI 3-K activity in eolae as reported for some G protein-coupled recep- C6 cells is modulated by P2Y and P2Y receptor tors (Bhatnagar et al. [81]). 1 12 expression. Another cross-talk at the level of PI 3-K/ Although experiments in CHO cells reveal that P2Y PKB is observed for P2Y and "-AR. Increase in receptor-induced ERK activation requires PI 3-K+ cAMP upon stimulation of the latter receptor tran- (Souletetal. [92]), experiments performed with siently inhibits PKB phosphorylation. Stimulation of LY294002 or Wortmannin excluded cross-talk between the P2Y receptor, which negatively affects AC, does both cascades in C6 cells (Grobben et al. [40]). These not only counteract this inhibition but even enhances differences in signalling mechanisms can be explained PKB activity in comparison to unstimulated cells, by the fact that the latter PI 3-K-isoform is only suggesting that P2Y receptor-mediated PI 3-K/PKB moderately expressed in C6 cells (Van Kolen and activation is not only due to its inhibitory effect on AC Slegers [45]). The exact mechanism of P2Y receptor- (Van Kolen and Slegers [45]; Czajkowski et al. [46]; induced PI 3-K/PKB activation is not fully understood, Baranska et al. [183]). In addition to their opposing but recent data revealed that Src and Pyk2 are involved effects on PI 3-K/PKB signalling, unpublished data of in P2Y receptor signalling to PI 3-K (Van Kolen et al., our laboratory revealed similar modulation of ERK [185]). A similar pathway is observed in PC12 cells signalling by P2Y and "-AR. Whether the P2Y where Src, in complex with Pyk2 and PLD2, activates 12 12 receptor-mediated reversal of ERK inhibition is in- PI 3-K in response to H O (Banno et al., [186]). Since 2 2 volved in the inhibition of "-AR-induced GFAP PLD2 is constitutively active in C6 cells (Bobeszko synthesis remains to be determined. The observation et al. [187]), a significant role for this enzyme in PI 3-K/ that stimulation of the cells with UTP activates ERK, Akt signalling is suggested. Although Soulet et al. [92] but fails to inhibit the "-AR-induced growth arrest and reported that transactivation of PDGFR is involved in GFAP synthesis, suggests that ERK activation alone is PI 3-K activation by the P2Y receptor in CHO cells, not sufficient to counteract differentiation (Claes et al. the use of receptor kinase inhibitors indicated that [39]; Tu et al. [44]). Conversely, transfection of C6 cells PDGFR and EGFR are not transactivated by the P2Y with constutively active PKB prevented (-)-isoproter- receptor in C6 cells. Alternatively, a Rap1-mediated enol-induced differentiation indicating that inhibition activation of PI 3-K by the P2Y receptor cannot be of PKB signalling is required for cAMP-dependent excluded. Indeed, PI 3-K is postulated as a downstream induction of differentiation. Apparently this observa- effector of Rap1 that is inhibited by an increase in tion is in contrast with data showing that cAMP- cAMP concentration (Wang et al. [149]). Data from our dependent induction of differentiation requires PI 3-K laboratory indicated a rapid P2Y receptor-induced 2+ activity which is not inhibited upon a 48-h treatment activation of Rap1 that was abolished by Ca chelation with dbcAMP (Roymans et al. [34]). This might be and inhibition of Src/Pyk2 complex formation but not explained by the fact that induction of differentiation by PI 3-K inhibition (Van Kolen et al. [185]). These by stimulation of "-AR proceeds through transient results positioned Rap1 downstream of Src/Pyk2 but Purinergic Signalling (2006) 2:451–469 463 upstream of PI 3-K. In addition, this mechanism determined by differential expression of signalling 2+ involves G"+ protein subunits and Ca -dependent proteins, but on the other hand also depends on the activation of Pyk2 that requires association to IGF-IR assembly of signalling modules. Besides specific pro- and PLD2 to interact with Src. Although Src and Pyk2 tein-protein interactions, intracellular compartmentali- are shown to activate Ras/Raf/MEK/ERK in primary sation (e.g., lipid rafts, clathrin-coated vesicles) also astrocytes (Wang and Reiser [83]), this mechanism did contributes to the specificity of receptor signalling. not contribute to P2Y receptor-mediated ERK acti- Identification of the signalling modules and cellular vation in C6 cells pointing to a physical separation of compartmentalisation will provide more insight into the both cascades (Grobben et al. [40]; Van Kolen and P2Y receptor-activated signalling cascades. Slegers, [199]). Indeed, the formation of a Pyk2/Src/ PLD2/IGFI-R complex may contribute to compartmen- Acknowledgment This work was supported by grants from the talisation of this signalling pathway that requires intact Fund for Scientific Research Flanders (HS) and BOF-NOI (HS). lipid rafts to be active (Van Kolen et al. [185]). 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Purinergic SignallingPubmed Central

Published: Jun 7, 2006

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